Abstract:

A system and method to test for the presence of target molecules in a
biological test sample includes test molecules, a microfluidic chip, and
irradiating and detection devices. The test molecules include
bio-recognition molecules conjugable with the target molecules, and the
corresponding conjugates. The microfluidic chip includes sample channels
and flow focusing channels adjoining the sample channels. A buffer
exiting from the focusing channels directs a single-file stream of the
test molecules through one of the sample channels. The irradiating device
delivers radiation for absorption by the test molecules in the
single-file stream. After absorption, the test molecules emit
fluorescence of a distinct fluorescent spectrum for each of the
conjugates. The detection device monitors identifies the presence of the
conjugates by monitoring for the distinct fluorescent spectrum. Thus, the
test system and method identifies the presence of the target molecules in
the test sample.

Claims:

1. A test system for use with a buffer to test for the presence of target
molecules of one or more target types in a biological test sample, the
test system comprising:(a) a first set of detection molecules, each
comprising one or more biorecognition molecules (BRMs) immobilized
relative to one or more BRM fluorophores, wherein each of the BRMs is
specific for one of the target types, so as to form conjugates of the
BRMs and the target molecules, if present in the test sample, with the
conjugates comprising one or more conjugate types each corresponding to a
different one of the detection molecules in conjugation with its specific
one of the target types;(b) a microfluidic chip comprising a chip
substrate portion shaped to define:(i) one or more elongate sample
channels therein sized to enable passage therethrough of the conjugates;
and(ii) one or more flow focusing channels therein for operative passage
therethrough of the buffer, with the one or more flowr focusing
channels adjoining the one or more elongate sample channels, with the
buffer exiting from the flow focusing channels operatively directing a
single-file stream of the conjugates through at least one of the sample
channels;(c) an irradiating device operatively delivering electromagnetic
frequency (EMF) radiation, at an irradiation position along said at least
one of the sample channels, for absorption by the conjugates in the
single-file stream, wherein the conjugates emit fluorescence after
absorption of the EMF radiation, and wherein the fluorescence of the
conjugates comprises a distinct fluorescent spectrum for each one of the
conjugate types; and(d) a detection device monitoring the single-file
stream for the fluorescence emitted by the conjugates, wherein the
detection device identifies the presence of the conjugates by monitoring
for the distinct fluorescent spectrum of each one of the conjugate
types;whereby the test system identifies the presence of the target
molecules in the test sample.

2. The test system according to claim 1, wherein each of the detection
molecules further comprises a microbead immobilized relative to, and
substantially interposed between, the BRMs and the BRM fluorophores, and
wherein the BRM fluorophores emit at least a BRM part of the fluorescence
of the distinct fluorescent spectrum after absorption of the EMF
radiation.

3. The test system according to claim 2, wherein the BRM fluorophores
comprise one or more quantum dots, with the quantum dots comprising one
or more quantum dot types, and wherein the quantum dots together emit at
least said BRM part of the fluorescence of the distinct fluorescent
spectrum after absorption of the EMF radiation.

4. The test system according to claim 3, wherein the quantum dots comprise
two or more of the quantum dot types.

5. The test system according to claim 2, wherein the BRM fluorophores
comprise one or more fluorescent dyes, with the fluorescent dyes
comprising one or more fluorescent dye types, and wherein the fluorescent
dyes together emit at least said BRM part of the fluorescence of the
distinct fluorescent spectrum after absorption of the EMF radiation.

6. The test system according to claim 1, wherein the conjugates are less
than about 10 micrometers (μm) in size.

7. The test system according to claim 6, wherein the conjugates are less
than about 5 μm in size.

8. The test system according to claim 7, wherein the conjugates are less
than about 1 μm in size.

9. The test system according to claim 1, wherein each of the conjugates
further comprises a target marker fluorophore bound to a respective one
of the target molecules, and wherein the target marker fluorophore emits
a target part of the fluorescence of the distinct fluorescent spectrum
after absorption of the EMF radiation.

10. The test system according to claim 1, wherein each of the detection
molecules further comprises a microbead immobilized relative to, and
substantially interposed between, the BRMs and the BRM fluorophores,
wherein each of the conjugates further comprises a target marker
fluorophore bound to a respective one of the target molecules, and
wherein for each of the conjugates, the BRM fluorophores emit a BRM part,
and the target marker fluorophore emits a target part, of the
fluorescence of the distinct fluorescent spectrum after absorption of the
EMF radiation, such that the BRM fluorophores and the target marker
fluorophore together emit the fluorescence of the distinct fluorescent
spectrum after absorption of the EMF radiation.

11. The test system according to claim 10, wherein the detection device
comprises at least two avalanche photodetectors (APDs) monitoring the
single-file stream for the fluorescence emitted by the conjugates, with a
first one of the APDs adapted to receive and identify the presence of the
BRM part, and a second one of the APDs adapted to receive and identify
the presence of the target part, of the fluorescence of the distinct
fluorescent spectrum for said each of the conjugates.

12. The test system according to claim 11, wherein the target part has a
lower intensity than the BRM part of the fluorescence of the distinct
fluorescent spectrum for said each of the conjugates, and wherein the
second one of the APDs has a greater sensitivity than the first one of
the APDs.

13. A test system to test for the presence of target molecules of one or
more target types in a biological test sample, with the test system being
for use with:a buffer; anda first set of detection molecules, with each
of the detection molecules comprising one or more biorecognition
molecules (BRMs) immobilized relative to one or more BRM fluorophores,
wherein each of the BRMs is specific for one of the target types, so as
to form conjugates of the BRMs and the target molecules, if present in
the test sample, with the conjugates comprising one or more conjugate
types each corresponding to a different one of the detection molecules in
conjugation with its specific one of the target types, with the detection
molecules emitting fluorescence after absorption of electromagnetic
frequency (EMF) radiation;wherein the test system comprises:(a) a
microfluidic chip comprising a chip substrate portion shaped to
define:(i) one or more elongate sample channels therein sized to enable
passage therethrough of the conjugates; and(ii) one or more flow focusing
channels therein for operative passage therethrough of the buffer, with
the one or more flow focusing channels adjoining the one or more elongate
sample channels, with the buffer exiting from the flow focusing channels
operatively directing a single-file stream of the conjugates through at
least one of the sample channels;(b) an irradiating device operatively
delivering the EMF radiation, at an irradiation position along said at
least one of the sample channels, for absorption by the conjugates in the
single-file stream, wherein the fluorescence of the conjugates comprises
a distinct fluorescent spectrum for each one of the conjugate types;
and(c) a detection device monitoring the single-file stream for the
fluorescence emitted by the conjugates, wherein the detection device
identifies the presence of the conjugates by monitoring for the distinct
fluorescent spectrum of each one of the conjugate types;whereby the test
system identifies the presence of the target molecules in the test
sample.

14. The test system according to claim 1, wherein said at least one of the
sample channels is defined by one or more elongate channel walls of the
chip substrate portion, with the channel walls comprising two opposing
side channel portions, and wherein the buffer exiting from the flow
focusing channels operatively directs the single-file stream of the
conjugates along a sample path that is in spaced relation from at least
the two opposing side channel portions.

15. The test system according to claim 14, wherein the microfluidic chip
further comprises a glass slide underlying the chip substrate portion,
with the glass slide defining a bottom channel portion of said at least
one of the sample channels, wherein the channel walls further comprise a
top channel portion, and wherein the sample path is operatively
positioned in said spaced relation from both the bottom channel portion
and the top channel portion.

16. The test system according to claim 1, wherein said at least one of the
sample channels comprises a sample focused channel, with the sample
channels further comprising a sample supply channel in fluid
communication with the sample focused channel, and with the sample
focused channel being downstream of the flow focusing channels, such that
the buffer exiting from the flow focusing channels and the single-file
stream of the conjugates operatively flow through the sample focused
channel.

17. The test system according to claim 16, wherein a buffer flow rate of
the buffer in the sample focused channel is higher than a test flow rate
of the conjugates in the single-file stream.

18. The test system according to claim 1, wherein the flow focusing
channels comprise at least two flow focusing channels, adjoining the one
or more elongate sample channels upstream of said at least one of the
sample channels, with the two flow focusing channels adjoining the one or
more elongate sample channels from opposing sides of said at least one of
the sample channels.

19. The test system according to claim 18, wherein the two flow focusing
channels adjoin the one or more elongate sample channels at a common
intersection portion.

20. The test system according to claim 19, wherein the buffer exiting from
the flow focusing channels operatively focuses the conjugates into the
single-file stream by less than about 10 micrometers (μm) downstream
of the common intersection portion.

21. The test system according to claim 1, wherein each of the one or more
flow focusing channels adjoins the one or more elongate sample channels
at an adjoining angle of about 90 degrees.

22. The test system according to claim 1, wherein each of the one or more
flow focusing channels adjoins the one or more elongate sample channels
at an adjoining angle of about 45 degrees.

23. The test system according to claim 1, wherein the chip substrate
portion is fabricated from polydimethylsiloxane (PDMS).

24. The test system according to claim 1, wherein passage of the
conjugates through said at least one of the sample channels is
facilitated by electrokinetic flow.

25. The test system according to claim 24, wherein the flow focusing
channels are in fluid communication with the sample channels; wherein the
chip substrate portion is further shaped to define a buffer well adjacent
a buffer starting point of each one of the flow focusing channels, a
sample well adjacent a sample starting point of the sample channels
upstream of the flow focusing channels, and a terminal well adjacent an
end point of said at least one of the sample channels downstream of the
flow focusing channels; wherein the test system further comprises a
sample well electrode operatively positioned in the sample well, a buffer
well electrode operatively positioned in each said buffer well, and a
terminal well electrode operatively positioned in the terminal well;
wherein the sample well electrode is operatively supplied with a first
electrical potential of a first polarity; wherein the terminal well
electrode is operatively supplied with a second electrical potential of
an opposing second polarity; and wherein each said buffer well electrode
is operatively supplied with a third electrical potential of the first
polarity.

26. The test system according to claim 25, wherein the third electrical
potential is higher than the first electrical potential.

27. The test system according to claim 26, wherein a ratio of the third
electrical potential relative to the first electrical potential is about
1.8:1.

28. The test system according to claim 1, wherein a test flow rate of the
conjugates in the single-file stream is at least about 30 conjugates per
minute.

29. The test system according to claim 28, wherein the test flow rate is
at least about 60 conjugates per minute.

30. The test system according to claim 29, wherein the test flow rate is
about 500 conjugates per minute.

31. A test system to test for the presence of target molecules of one or
more target types in a biological test sample, with the test system being
for use with: a first set of detection molecules, each comprising one or
more biorecognition molecules (BRMs) immobilized relative to one or more
BRM fluorophores, wherein each of the BRMs is specific for one of the
target types, so as to form conjugates of the BRMs and the target
molecules, if present in the test sample, with the conjugates comprising
one or more conjugate types each corresponding to a different one of the
detection molecules in conjugation with its specific one of the target
types, with the conjugates emitting fluorescence after absorption of
electromagnetic frequency (EMF) radiation; and a microfluidic chip
comprising a chip substrate portion shaped to define one or more elongate
sample channels therein sized to enable passage therethrough of the
conjugates, with a single-file stream of the conjugates passing through
at least one of the sample channels;wherein the test system comprises:(a)
an irradiating device operatively delivering the EMF radiation, at an
irradiation position along said at least one of the sample channels, for
absorption by the conjugates in the single-file stream, wherein the
conjugates emit fluorescence after absorption of the EMF radiation, and
wherein the fluorescence of the conjugates comprises a distinct
fluorescent spectrum for each one of the conjugate types; and(b) a
detection device monitoring the single-file stream for the fluorescence
emitted by the conjugates, wherein the detection device identifies the
presence of the conjugates by monitoring for the distinct fluorescent
spectrum of each one of the conjugate types;whereby the test system
identifies the presence of the target molecules in the test sample.

32. The test system according to claim 1, wherein the irradiating device
comprises a light emitting diode (LED) operatively emitting the EMF
radiation for absorption by the conjugates in the single-file stream.

33. The test system according to claim 1, wherein the irradiating device
comprises a laser operatively emitting the EMF radiation for absorption
by the conjugates in the single-file stream.

34. The test system according to claim 33, wherein the laser has an
operating power of between about 2 megawatts (mW) and about 50 megawatts
(mW).

35. The test system according to claim 34, wherein the operating power of
the laser is between about 20 megawatts (mW) and about 25 megawatts (mW).

36. The test system according to claim 1, wherein the EMF radiation
operatively delivered by the irradiating device has an EMF wavelength of
about 488 nm.

37. The test system according to claim 1, wherein the detection device
comprises three or more avalanche photodetectors (APDs) monitoring the
single-file stream for the fluorescence emitted by the conjugates, with
each of the APDs adapted to receive and identify the presence of a
different range of wavelengths in the fluorescence emitted by the
conjugates.

38. The test system according to claim 37, wherein a first one of the APDs
is adapted to receive and identify the presence of a green range of
wavelengths, wherein a second one of the APDs is adapted to receive and
identify the presence of a yellow range of wavelengths, and wherein a
third one of the APDs is adapted to receive and identify the presence of
a red range of wavelengths.

39. The test system according to claim 37, wherein a first one of the APDs
is adapted to receive and identify the presence of a green range of
wavelengths, wherein a second one of the APDs is adapted to receive and
identify the presence of a yellow range of wavelengths, wherein a third
one of the APDs is adapted to receive and identify the presence of an
orange range of wavelengths, and wherein a fourth one of the APDs is
adapted to receive and identify the presence of a red range of
wavelengths.

40. The test system according to claim 37, wherein a first one of the APDs
is adapted to receive and identify the presence of a blue range of
wavelengths, wherein a second one of the APDs is adapted to receive and
identify the presence of a green range of wavelengths, wherein a third
one of the APDs is adapted to receive and identify the presence of a
yellow range of wavelengths, and wherein a fourth one of the APDs is
adapted to receive and identify the presence of a red range of
wavelengths.

41. The test system according to claim 1, wherein the detection device
comprises a charge-coupled device monitoring the single-file stream for
the fluorescence emitted by the conjugates.

42. The test system according to claim 1, wherein the detection device
comprises at least two avalanche photodetectors (APDs) monitoring the
single-file stream for the fluorescence emitted by the conjugates, with
each of the APDs adapted to receive and identify the presence of a
different range of wavelengths in the fluorescence emitted by the
conjugates; wherein the detection device further comprises a
charge-coupled device monitoring the single-file stream for the
fluorescence emitted by the conjugates; and wherein the detection device
still further comprises a switch means for switching between monitoring
the single-file stream with either the APDs or the charge-coupled device.

43. The test system according to claim 1, wherein the detection device
comprises at least one trip sensor monitoring the single-file stream for
the fluorescence emitted by the conjugates, with each said trip sensor
generating a digital signal corresponding to an intensity of the
fluorescence.

44. The test system according to claim 43, wherein each said trip sensor
generates the digital signal only when the intensity of the fluorescence
is in excess of a minimum intensity, and wherein each said trip sensor
has a different pre-determined said minimum intensity.

45. The test system according to claim 1, further comprising a fiber optic
cable, with the fiber optic cable operatively delivering the fluorescence
to the detection device from substantially adjacent to the irradiation
position along said at least one of the sample channels.

46. The test system according to claim 1, further comprising a housing
encasing the irradiating device and the detection device, with the
housing sized and adapted for portable and point-of-care diagnostic use.

47. The test system according to claim 46, wherein the housing is sized
and adapted for hand-held use.

48. A test system to test for the presence of target molecules of one or
more target types in a biological test sample, with the test system being
for use with:a buffer;a first set of detection molecules, each comprising
one or more biorecognition molecules (BRMs) immobilized relative to one
or more BRM fluorophores, wherein each of the BRMs is specific for one of
the target types, so as to form conjugates of the BRMs and the target
molecules, if present in the test sample, with the conjugates comprising
one or more conjugate types each corresponding to a different one of the
detection molecules in conjugation with its specific one of the target
types; andan irradiating and detection device capable of delivering
electromagnetic frequency (EMF) radiation for absorption by the
conjugates, with the conjugates emitting fluorescence after absorption of
the EMF radiation, wherein the fluorescence of the conjugates includes a
distinct fluorescent spectrum for each one of the conjugate types, and
with the irradiation and detection device also capable of monitoring for
and identifying the conjugates by the presence of the distinct
fluorescent spectrum for each one of the conjugate types;wherein the test
system comprises a microfluidic chip comprising a chip substrate portion
shaped to define:(a) one or more elongate sample channels therein sized
to enable passage therethrough of the conjugates; and(b) one or more flow
focusing channels therein for operative passage therethrough of the
buffer, with the one or more flow focusing channels adjoining the one or
more elongate sample channels, with the buffer exiting from the flow
focusing channels operatively directing a single-file stream of the
conjugates through at least one of the sample channels;wherein the
microfluidic chip is adapted to operatively receive the EMF radiation
from the irradiating and detection device, at an irradiation position
along said at least one of the sample channels, for absorption by the
conjugates in the single-file stream; andwherein the microfluidic chip is
adapted to enable the irradiation and detection device to monitor the
single-file stream for the fluorescence emitted by the conjugates;whereby
the conjugates are operatively identifiable by the presence of the
distinct fluorescent spectrum for each one of the conjugate types, such
that the presence of the target molecules in the test sample is
operatively identifiable by the test system.

49. The test system according to claim 48, wherein said at least one of
the sample channels is defined by one or more elongate channel walls of
the chip substrate portion, with the channel walls comprising two
opposing side channel portions, and wherein the buffer exiting from the
flow focusing channels operatively directs the single-file stream of the
conjugates along a sample path that is in spaced relation from at least
the two opposing side channel portions,

50. The test system according to claim 49, wherein the microfluidic chip
further comprises a glass slide underlying the chip substrate portion,
with the glass slide defining a bottom channel portion of said at least
one of the sample channels, wherein the channel walls further comprise a
top channel portion, and wherein the sample path is operatively
positioned in said spaced relation from both the bottom channel portion
and the top channel portion.

51. The test system according to claim 48, wherein said at least one of
the sample channels comprises a sample focused channel, with the sample
channels further comprising a sample supply channel in fluid
communication with the sample focused channel, and with the sample
focused channel being downstream of the flow focusing channels, such that
the buffer exiting from the flow focusing channels and the single-file
stream of the conjugates operatively flow through the sample focused
channel.

52. The test system according to claim 48, wherein the flow focusing
channels comprise at least two flow focusing channels, adjoining the one
or more elongate sample channels upstream of said at least one of the
sample channels, with the two flow focusing channels adjoining the one or
more elongate sample channels from opposing sides of said at least one of
the sample channels.

53. The test system according to claim 52, wherein the two flow focusing
channels adjoin the one or more elongate sample channels at a common
intersection portion.

54. The test system according to claim 53, wherein the buffer exiting from
the flow focusing channels operatively focuses the conjugates into the
single-file stream by less than about 10 micrometers (μm) downstream
of the common intersection portion.

55. The test system according to claim 48, wherein each of the one or more
flow focusing channels adjoins the one or more elongate sample channels
at an adjoining angle of about 90 degrees.

56. The test system according to claim 48, wherein each of the one or more
flow focusing channels adjoins the one or more elongate sample channels
at an adjoining angle of about 45 degrees.

57. The test system according to claim 48, wherein the chip substrate
portion is fabricated from polydimethylsiloxane (PDMS).

58. The test system according to claim 48, wherein passage of the
conjugates through said at least one of the sample channels is
facilitated by electrokinetic flow.

59. The test system according to claim 58, wherein the flow focusing
channels are in fluid communication with the sample channels; wherein the
chip substrate portion is further shaped to define a buffer well adjacent
a buffer starting point of each one of the flow focusing channels, a
sample well adjacent a sample starting point of the sample channels
upstream of the flow focusing channels, and a terminal well adjacent an
end point of said at least one of the sample channels downstream of the
flow focusing channels; wherein the test system further comprises a
sample well electrode operatively positioned in the sample well, a buffer
well electrode operatively positioned in each said buffer well, and a
terminal well electrode operatively positioned in the terminal well;
wherein the sample well electrode is operatively supplied with a first
electrical potential of first polarity; wherein the terminal well
electrode is operatively supplied with a second electrical potential of
an opposing second polarity; and wherein each said buffer well electrode
is operatively supplied with a third electrical potential of the first
polarity.

60. The test system according to claim 59, wherein the third electrical
potential is higher than the first electrical potential.

61. The test system according to claim 60, wherein a ratio of the third
electrical potential relative to the first electrical potential is about
1.8:1.

62. The test system according to claim 61, wherein a test flow rate of the
conjugates in the single-file stream is at least about 30 conjugates per
minute.

63. The test system according to claim 62, wherein the test flow rate is
at least about 60 conjugates per minute.

64. The test system according to claim 63, wherein the test flow rate is
about 500 conjugates per minute.

65. The test system according to claim 1, wherein a buffer flow rate of
the buffer in the sample focused channel is higher than a test flow rate
of the conjugates in the single-file stream.

66. The test system according to claim 1, wherein the test system is
particularly adapted for use with one or more biological test samples
selected from the group consisting of blood, urine, sputum, and serum.

67. Use of the test system of claim 1 for a diagnosis of a disease state
selected from the group consisting of bacterial disease states, viral
disease states, fungal disease states, and vector-induced disease states.

68. Use of the test system of claim 1 for a diagnosis of one or more
infectious diseases.

69. Use of the test system of claim 1 for a diagnosis of a condition
selected from the group consisting of HIV, HBV and HCV.

70. Use of the test system of claim 1 for a simultaneous diagnosis of two
or more of the conditions selected from the group consisting of HIV HBV
and HCV.

71. A method of facilitating a test for the presence of target molecules
of one or more target types in a biological test sample, the method
comprising the steps of:a sample flowing step of passing detection
molecules through one or more elongate sample channels formed in a chip
substrate portion of a microfluidic chip, wherein each of the detection
molecules comprises one or more biorecognition molecules (BRMs)
immobilized relative to one or more BRM fluorophores, wherein each of the
BRMs is specific for one of the target types, so as to form conjugates of
the BRMs and the target molecules, if present in the test sample, with
the conjugates comprising one or more conjugate types each corresponding
to a different one of the detection molecules in conjugation with its
specific one of the target types;a buffer flowing step of passing a
buffer through one or more flow focusing channels formed in the chip
substrate portion of the microfluidic chip, with the flow focusing
channels adjoining the one or more elongate sample channels; anda sample
focusing step, after the buffer flowing step, of directing a single-file
stream of the conjugates through at least one of the sample channels by
passage of the buffer from the flow focusing channels into the one or
more elongate sample channels.

72. The method according to claim 71, further comprising a
conjugate-forming step, before the sample flowing step, of forming the
conjugates by introducing the BRMs into the test sample, so as to form
the conjugates of the BRMs and the target molecules, if present in the
test sample.

73. The method according to claim 72 wherein, in the conjugate-forming
step, the conjugates are of less than about 10 micrometers (μm) in
size.

74. The method according to claim 73 wherein, in the conjugate-forming
step, the conjugates are of less than about 5 micrometers (μm) in
size.

75. The method according to claim 74 wherein, in the conjugate-forming
step, the conjugates are of less than about 1 micrometer (μm) in size.

76. The method according to claim 72 wherein, in the conjugate-forming
step, target marker fluorophores conjugable with one or more of the
target types are introduced, such that the target marker fluorophores are
immobilized relative to and the conjugates.

77. The method according to claim 71 wherein, in the sample focusing step,
the single-file stream of the conjugates is directed along a sample path
that is in spaced relation from at least two opposing side channel
portions of said at least one of the sample channels.

78. The method according to claim 71 wherein, in the sample focusing step,
the single-file stream of the conjugates is directed along a sample path
that is in spaced relation from at least top and bottom channel portions
of said at least one of the sample channels.

79. The method according to claim 71 wherein, in the sample focusing step,
the buffer flows into said at least one of the sample channels at a
buffer flow rate that is higher than a test flow rate of the conjugates
in the single-file stream.

80. The method according to claim 71 wherein, in the sample focusing step,
at least two of the flow focusing channels adjoin the sample channels,
from opposing sides thereof, upstream of said at least one of the sample
channels.

81. The method according to claim 80 wherein, in the sample focusing step,
the two flow focusing channels adjoin the sample channels at a common
intersection portion.

82. The method according to claim 71 wherein, in the sample focusing step,
each of the one or more flow focusing channels adjoins the sample
channels at an adjoining angle of about 90 degrees.

83. The method according to claim 71 wherein, in the sample focusing step,
each of the one or more flow focusing channels adjoins the sample
channels at an adjoining angle of about 45 degrees.

84. The method according to claim 71 wherein, in the sample focusing step,
passage of the single-file stream of the conjugates through said at least
one of the sample channels is facilitated by electrokinetic flow.

85. The method according to claim 84, further comprising an electrokinetic
step, before the sample focusing step, of supplying (i) a first
electrical potential of a first polarity to the sample channels upstream
of the flow focusing channels, (ii) a second electrical potential of an
opposing second polarity to said at least one of the sample channels
downstream of the flow focusing channels, and (iii) a third electrical
potential of the first polarity to each one of the flow focusing
channels.

86. The method according to claim 85 wherein, in the electrokinetic step,
the third electrical potential is higher than the first electrical
potential.

87. The method according to claim 86 wherein, in the electrokinetic step,
a ratio of the third electrical potential relative to the first
electrical potential is about 1.8:1.

88. The method according to claim 72 wherein the method further comprises
the steps of:an irradiating step, after the sample focusing step, of
delivering electromagnetic frequency (EMF) radiation to the conjugates in
the single-file stream; anda fluorescence-detecting step, after the
irradiating step, of monitoring the single-file stream for fluorescence
emitted by the conjugates, with each of the conjugates, after absorption
of the EMF radiation, emitting fluorescence of a distinct fluorescent
spectrum for each one of the conjugate types; anda conjugate-identifying
step, after the irradiating step, of monitoring for the distinct
fluorescent spectrum of each one of the conjugate types, so as to
identify the presence of the target molecules in the test sample.

89. The method according to claim 88 wherein, in the conjugate-forming
step, target marker fluorophores are bound to respective ones of the
target molecules, such that in the fluorescence-detecting step, the
target marker fluorophores emit a target part, and the BRM fluorophores
emit a BRM part, of the distinct fluorescent spectrum for each one of the
conjugate types.

90. The method according to claim 89 wherein, in the
fluorescence-detecting step, fluorescence emitted by the conjugates is
received by at least two avalanche photodetectors (APDs), with a first
one of the APDs receiving and identifying the presence of the BRM part,
and a second one of the APDs receiving and identifying the presence of
the target part, of the fluorescence of the distinct fluorescent spectrum
for said each of the conjugates.

91. The method according to claim 88 wherein, in the irradiating step, a
laser having an operating power of between about 2 megawatts (mW) and
about 50 megawatts (mW) delivers the EMF radiation to the conjugates in
the single-file stream.

92. The method according to claim 91 wherein, in the irradiating step, the
operating power is between about 20 megawatts (mW) and about 25 megawatts
(mW).

93. The method according to claim 88 wherein, in the irradiating step, the
EMF radiation has an EMF wavelength of about 488 nm.

94. The method according to claim 88 wherein, in the
fluorescence-detecting step, the fluorescence emitted by the conjugates
is received by a charge-coupled device.

95. The method according to claim 88 wherein, in the
fluorescence-detecting step, the fluorescence emitted by the conjugates
is selectively received by at least one of a charge-coupled device and
one or more avalanche photodetectors (APDs).

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to the field of microfluidics. In
particular, it relates to a microfluidic channel structure for reading
fluorescent microbeads.

BACKGROUND OF THE INVENTION

[0002]The last decade has seen many advances in the fields of
microtechnology and nanotechnology. One of the challenges created by
these advances is developing practical uses for discovered scientific
phenomena.

[0003]A few published reports of attempts to integrate nano- with
microtechnology for biomolecular or viral detection have been described
[W. Liu et al., Lab Chip, 5, 1327 (2005); K. Yun, D. Lee, H. Kim, E.
Yoon, Meas. Sci. Technol., 17, 3178 (2006); J. Steigert et al., JALA, 10,
331 (2005)]. In these studies, the researchers used a combination of
nanoparticles, microbeads, and microfluidics for detection. In all cases,
the detection sensitivity was lower than desirable for a productive,
commercial product. Furthermore, the analysis was not conducted in serum,
which could decrease sensitivity because of interference from blood
components [E. D. Goluch et al., Lab Chip, 6, 1293 (2006)].

[0004]Similarly, bio-barcodes using gold nanoparticles have been
demonstrated for applications in genomic or proteomic diagnostics [J.
Tate, G. Ward, Clin. Biochem. Rev., 25, 105 (2004); S. I. Stoeva, J. Lee,
C. S. Thaxton, C. A. Mirkin, Angew. Chem. Int. Ed., 45, 3303 (2006); P.
Mitchell, Nat. Biotech., 20, 225 (2002)]). In these methods, the
detection strategy requires multiple steps to achieve assay detection as
well as amplification to achieve good sensitivity. Thus, there is a need
for a detection system that only requires a few steps and can achieve a
reasonably high level of sensitivity.

[0005]Published United States Patent Application No. US2007/0020779 of
Stavis et al. discloses a method of detecting quantum dots conjugates in
a sub-micrometer fluidic channel. The cross-sectional size of the
channels used in Stavis is on the order of 500 nm and the detected
conjugates on the order of 5-10 nm. Furthermore, in order to achieve
single conjugate detection, the concentration of the sample was reduced
to the femtomolar level, increasing the difficulty of sample preparation
and the limits on the detection system. An alternative and more efficient
system and method of single conjugate detection, ideally for use with
more easily handled micro-scale molecules, is needed.

[0006]Objects of this invention are preferably accomplished, but may not
be necessarily as described, nor is it necessary for all objects to be
accomplished by a single embodiment of the invention. Additional objects
may be accomplished that are not listed herein.

[0007]It is an object of this invention to enable multiplexed detection of
target molecules of one or more target types by irradiating and detecting
fluorescent emission from a single-file stream of test molecules.

[0008]It is an object of this invention to enable testing of biological
samples for infectious diseases. It is a further object to enable testing
of specific biological samples of blood, serum, sputum and/or urine.

[0009]It is an object of this invention to enable multiplexed testing for
infectious diseases in biological samples. It is a further object to
enable multiplexed testing for Hepatitis B, Hepatitis C and HIV in any
combination.

[0010]It is an object of this invention to provide an improved
microfluidic channel structure that facilitates flow through the
channels.

[0011]It is an object of this invention to provide a fixed-wavelength EMF
radiation device, such as a 488 nm laser, as the irradiation device in a
test system such that the incident EMF radiation and emitted fluorescence
from the target molecule can travel along the same optical path prior to
the emitted fluorescence entering the detection device.

[0012]It is an object of this invention to partially or completely fulfill
one or more of the above-mentioned objects and to mitigate and/or
ameliorate any disadvantages of the prior art, regardless of whether any
such disadvantages are described herein.

SUMMARY OF THE INVENTION

[0013]In accordance with the present invention there is disclosed a test
system for use with a buffer to test for the presence of target molecules
of one or more target types in a biological test sample. The test system
includes a first set of test molecules, a microfluidic chip, an
irradiating device, and a detection device. The first set of test
molecules is selected from a group that includes bio-recognition
molecules (BRMs) and conjugates of the BRMs and the target molecules, if
present in the test sample. The BRMs are of one or more BRM types. Each
of the BRM types is conjugable with a respective one of the target types.
The conjugates are of one or more conjugate types each corresponding to a
different one of the BRM types in conjugation with its said respective
one of the target types. The microfluidic chip includes a chip substrate
portion that is shaped to define one or more elongate sample channels,
and one or more flow focusing channels, therein. The sample channels are
sized to enable passage therethrough of the test molecules. The flow
focusing channels are for operative passage therethrough of the buffer.
The one or more flow focusing channels adjoin the one or more elongate
sample channels. The buffer exits from the flow focusing channels and
operatively directs a single-file stream of the test molecules through at
least one of the sample channels. The irradiating device operatively
delivers electromagnetic frequency (EMF) radiation, at an irradiation
position along the aforesaid at least one of the sample channels, for
absorption by the test molecules in the single-file stream. The test
molecules emit fluorescence after absorption of the EMF radiation. The
fluorescence of the test molecules includes a distinct fluorescent
spectrum for each one of the conjugate types. The detection device
monitors the single-file stream for the fluorescence emitted by the test
molecules. The detection device identifies the presence of the conjugates
in the first set of test molecules by monitoring for the distinct
fluorescent spectrum of each one of the conjugate types. In this manner,
the test system identifies the presence of the target molecules in the
test sample.

[0014]According to an aspect of one preferred embodiment of the invention,
each of the BRMs includes a microbead tagged with one or more BRM
fluorophores that are coupled to the microbead. The BRM fluorophores emit
at least a BRM part of the fluorescence of the distinct fluorescent
spectrum after absorption of the EMF radiation.

[0015]According to an aspect of one preferred embodiment of the invention,
the BRM fluorophores include one or more quantum dots of one or more
quantum dot types. The quantum dots together emit at least the BRM part
of the fluorescence of the distinct fluorescent spectrum after absorption
of the EMF radiation.

[0016]According to an aspect of one preferred embodiment of the invention,
the quantum dots are of two or more of the quantum dot types.

[0017]According to an aspect of one preferred embodiment of the invention,
the BRM fluorophores include one or more fluorescent dyes of one or more
fluorescent dye types. The fluorescent dyes together emit at least the
BRM part of the fluorescence of the distinct fluorescent spectrum after
absorption of the EMF radiation.

[0018]According to an aspect of one preferred embodiment of the invention,
the conjugates are less than about 10 micrometers (μm) in size, and
preferably less than about 5 μm in size, and still more preferably,
less than about 1 μm in size.

[0019]According to an aspect of one preferred embodiment of the invention,
each of the conjugates further includes a target marker fluorophore bound
to a respective one of the target molecules. The target marker
fluorophore emits a target part of the fluorescence of the distinct
fluorescent spectrum after absorption of the EMF radiation.

[0020]According to an aspect of one preferred embodiment of the invention,
each of the BRMs includes a microbead tagged with one or more BRM
fluorophores that are coupled to the microbead. Each of the conjugates
further includes a target marker fluorophore bound to a respective one of
the target molecules. For each of the conjugates, the BRM fluorophores
emit a BRM part, and the target marker fluorophore emits a target part,
of the fluorescence of the distinct fluorescent spectrum after absorption
of the EMF radiation. As such, the BRM fluorophores and the target marker
fluorophore together emit the fluorescence of the distinct fluorescent
spectrum after absorption of the EMF radiation.

[0021]According to an aspect of one preferred embodiment of the invention,
the detection device includes at least two avalanche photodetectors
(APDs) monitoring the single-file stream for the fluorescence emitted by
the test molecules. A first one of the APDs is adapted to receive and
identify the presence of the BRM part, and a second one of the APDs
adapted to receive and identify the presence of the target part, of the
fluorescence of the distinct fluorescent spectrum for said each of the
conjugates.

[0022]According to an aspect of one preferred embodiment of the invention,
the target part has a lower intensity than the BRM part of the
fluorescence of the distinct fluorescent spectrum for each of the
conjugates. The second one of the APDs has a greater sensitivity than the
first one of the APDs.

[0023]In accordance with the present invention there is disclosed another
test system for use with a buffer to test for the presence of target
molecules of one or more target types in a biological test sample.
According to this embodiment of the invention, the test system is also
for use with a first set of test molecules selected from a group that
includes bio-recognition molecules (BRMs) and conjugates of the BRMs and
the target molecules, if present in the test sample. The BRMs are of one
or more BRM types. Each of the BRM types is conjugable with a respective
one of the target types. The test molecules are such as to emit
fluorescence after absorption of EMF radiation. The conjugates are of one
or more conjugate types each corresponding to a different one of the BRM
types in conjugation with its said respective one of the target types.
According to this embodiment of the invention, the test system includes a
microfluidic chip, an irradiating device, and a detection device. The
microfluidic chip includes a chip substrate portion shaped to define one
or more elongate sample channels, and one or more flow focusing channels,
therein. The sample channels are sized to enable passage therethrough of
the test molecules. The flow focusing channels are for operative passage
therethrough of the buffer. The flow focusing channels adjoin the sample
channels. The buffer exits from the flow focusing channels operatively
directing a single-file stream of the test molecules through at least one
of the sample channels. The irradiating device operatively delivers
electromagnetic frequency (EMF) radiation, at an irradiation position
along that aforesaid at least one of the sample channels, for absorption
by the test molecules in the single-file stream. The fluorescence of the
test molecules includes a distinct fluorescent spectrum for each one of
the conjugate types. The detection device monitors the single-file stream
for the fluorescence emitted by the test molecules. The detection device
identifies the presence of the conjugates in the first set of test
molecules by monitoring for the distinct fluorescent spectrum of each one
of the conjugate types. In this manner, the test system identifies the
presence of the target molecules in the test sample.

[0024]According to an aspect of one preferred embodiment of the invention,
said at least one of the sample channels is defined by one or more
elongate channel walls of the chip substrate portion. The channel walls
include two opposing side channel portions. The buffer, exiting from the
flow focusing channels, operatively directs the single-file stream of the
test molecules along a sample path that is in spaced relation from at
least the aforesaid two opposing side channel portions.

[0025]According to an aspect of one preferred embodiment of the invention,
the microfluidic chip further includes a glass slide underlying the chip
substrate portion. The glass slide defines a bottom channel portion of
said at least one of the sample channels. The channel walls additionally
include a top channel portion. The sample path is operatively in the
aforesaid spaced relation from both the bottom channel portion and the
top channel portion.

[0026]According to an aspect of one preferred embodiment of the invention,
the aforesaid at least one of the sample channels includes a sample
focused channel. The sample channels also include a sample supply channel
in fluid communication with the sample focused channel. The sample
focused channel is downstream of the flow focusing channels. As such, the
buffer exiting from the flow focusing channels and the single-file stream
of the test molecules operatively flow through the sample focused
channel.

[0027]According to an aspect of one preferred embodiment of the invention,
a buffer flow rate of the buffer, operatively flowing through the sample
focused channel, is higher than a test flow rate of the test molecules in
the single-file stream.

[0028]According to an aspect of one preferred embodiment of the invention,
the flow focusing channels include at least two flow focusing channels,
adjoining the sample channels upstream of the aforesaid at least one of
the sample channels. The two flow focusing channels adjoin the sample
channels from opposing sides of the aforesaid at least one of the sample
channels.

[0029]According to an aspect of one preferred embodiment of the invention,
the two flow focusing channels adjoin the sample channels at a common
intersection portion.

[0030]According to an aspect of one preferred embodiment of the invention,
the buffer exiting from the flow focusing channels operatively focuses
the test molecules into the single-file stream by less than about 10
micrometers (μm) downstream of the common intersection portion.

[0031]According to an aspect of one preferred embodiment of the invention,
each of the flow focusing channels adjoins the sample channels at an
adjoining angle of about 90 degrees.

[0032]According to an aspect of another preferred embodiment of the
invention, each of flow focusing channels adjoins the sample channels at
an adjoining angle of about 45 degrees.

[0033]According to an aspect of one preferred embodiment of the invention,
the chip substrate portion is fabricated from polydimethylsiloxane
(PDMS).

[0034]According to an aspect of one preferred embodiment of the invention,
passage of the test molecules through the aforesaid at least one of the
sample channels is facilitated by electrokinetic flow.

[0035]According to an aspect of one preferred embodiment of the invention,
the flow focusing channels are in fluid communication with the sample
channels. The chip substrate portion is additionally shaped to define a
buffer well, a sample well, and a terminal well. Each buffer well is
adjacent to a buffer starting point of each one of the flow focusing
channels. The sample well is adjacent to a sample starting point of the
sample channels upstream of the flow focusing channels. The terminal well
is adjacent to an end point of the aforesaid at least one of the sample
channels downstream of the flow focusing channels. The test system also
includes a sample well electrode, a buffer well electrode, and a terminal
well electrode. The sample well electrode is operatively positioned in
the sample well. Each buffer well electrode is operatively positioned in
one aforesaid buffer well. The terminal well electrode is operatively
positioned in the terminal well. The sample well electrode is operatively
supplied with a first electrical potential of a first polarity. The
terminal well electrode is operatively supplied with a second electrical
potential of an opposing second polarity. Each buffer well electrode is
operatively supplied with a third electrical potential of the first
polarity.

[0036]According to an aspect of one preferred embodiment of the invention,
the third electrical potential is higher than the first electrical
potential.

[0037]According to an aspect of one preferred embodiment of the invention,
a ratio of the third electrical potential relative to the first
electrical potential is about 1.8:1 (9:5).

[0038]According to an aspect of one preferred embodiment of the invention,
a test flow rate of the test molecules in the single-file stream is at
least about 30 test molecules per minute, and preferably at least about
60 test molecules per minute, and still more preferably about 500 test
molecules per minute.

[0039]In accordance with the present invention there is also disclosed a
further test system to test for the presence of target molecules of one
or more target types in a biological test sample. According to this
embodiment of the invention, the test system is for use with a first set
of test molecules selected from a group that includes bio-recognition
molecules (BRMs) and conjugates of the BRMs and the target molecules, if
present in the test sample. The BRMs are of one or more BRM types. Each
of the BRM types is conjugable with a respective one of the target types.
The conjugates are of one or more conjugate types, each corresponding to
a different one of the BRM types in conjugation with its aforesaid
respective one of the target types. The test system is also for use with
a microfluidic chip that includes a chip substrate portion, which is
shaped to define one or more elongate sample channels therein. The sample
channels are sized to enable passage therethrough of the test molecules.
A single-file stream of the test molecules passes through at least one of
the sample channels. According to this embodiment of the invention, the
test system includes an irradiating device and a detection device. The
irradiating device operatively delivers electromagnetic frequency (EMF)
radiation, at an irradiation position along the aforesaid at least one of
the sample channels, for absorption by the test molecules in the
single-file stream. The test molecules emit fluorescence after absorption
of the EMF radiation. The fluorescence of the test molecules includes a
distinct fluorescent spectrum for each one of the conjugate types. The
detection device monitors the single-file stream for the fluorescence
emitted by the test molecules. The detection device identifies the
presence of the conjugates in the first set of test molecules by
monitoring for the distinct fluorescent spectrum of each one of the
conjugate types. In this manner, the test system identifies the presence
of the target molecules in the test sample.

[0040]According to an aspect of one preferred embodiment of the invention,
the irradiating device includes an LED which operatively emits the EMF
radiation for absorption by the test molecules in the single-file stream.

[0041]According to an aspect of another preferred embodiment of the
invention, the irradiating device includes a laser, which operatively
emits the EMF radiation for absorption by the test molecules in the
single-file stream.

[0042]According to an aspect of one preferred embodiment of the invention,
the laser has an operating power of between about 2 megawatts (mW) and
about 50 megawatts (mW), and more preferably, between about 20 megawatts
(mW) and about 25 megawatts (mW).

[0043]According to an aspect of one preferred embodiment of the invention,
the EMF radiation operatively delivered by the irradiating device has an
EMF wavelength of about 488 nm.

[0044]According to an aspect of one preferred embodiment of the invention,
the detection device includes at least three avalanche photodetectors
(APDs) monitoring the single-file stream for the fluorescence emitted by
the test molecules. Each of the APDs is adapted to receive and identify
the presence of a different range of wavelengths in the fluorescence
emitted by the test molecules.

[0045]According to an aspect of one preferred embodiment of the invention,
a first one of the APDs is adapted to receive and identify the presence
of a green range of wavelengths. A second one of the APDs is adapted to
receive and identify the presence of a yellow range of wavelengths. A
third one of the APDs is adapted to receive and identify the presence of
a red range of wavelengths.

[0046]According to an aspect of another preferred embodiment of the
invention, the aforesaid at least three APDs include at least four APDs.
A first one of the APDs is adapted to receive and identify the presence
of a green range of wavelengths. A second one of the APDs is adapted to
receive and identify the presence of a yellow range of wavelengths. A
third one of the APDs is adapted to receive and identify the presence of
an orange range of wavelengths. A fourth one of the APDs is adapted to
receive and identify the presence of a red range of wavelengths.

[0047]According to an aspect of yet another preferred embodiment of the
invention, the at least three APDs include at least four APDs. A first
one of the APDs is adapted to receive and identify the presence of a blue
range of wavelengths. A second one of the APDs is adapted to receive and
identify the presence of a green range of wavelengths. A third one of the
APDs is adapted to receive and identify the presence of a yellow range of
wavelengths. A fourth one of the APDs is adapted to receive and identify
the presence of a red range of wavelengths.

[0048]According to an aspect of one preferred embodiment of the invention,
the detection device includes a charge-coupled device monitoring the
single-file stream for the fluorescence emitted by the test molecules.

[0049]According to an aspect of one preferred embodiment of the invention,
the detection device includes at least two avalanche photodetectors
(APDs) monitoring the single-file stream for the fluorescence emitted by
the test molecules. Each of the APDs is adapted to receive and identify
the presence of a different range of wavelengths in the fluorescence
emitted by the test molecules. The detection device additionally includes
a charge-coupled device monitoring the single-file stream for the
fluorescence emitted by the test molecules. Still further, the detection
device includes a switch means for switching between monitoring the
single-file stream with either the APDs or the charge-coupled device.

[0050]According to an aspect of one preferred embodiment of the invention,
the detection device includes at least one trip sensor monitoring the
single-file stream for the fluorescence emitted by the test molecules.
Each aforesaid trip sensor generates a digital signal corresponding to an
intensity of the fluorescence.

[0051]According to an aspect of one preferred embodiment of the invention,
each aforesaid trip sensor generates the digital signal only when the
intensity of the fluorescence is in excess of a minimum intensity. Each
aforesaid trip sensor has a different pre-determined said minimum
intensity.

[0052]According to an aspect of one preferred embodiment of the invention,
the test system also includes a fiber optic cable delivering the
fluorescence to the detection device from substantially adjacent to the
irradiation position along the aforesaid at least one of the sample
channels.

[0053]According to an aspect of one preferred embodiment of the invention,
the test system also includes a housing encasing the irradiating device
and the detection device. The housing is sized and adapted for portable
and point-of-care diagnostic use.

[0054]According to an aspect of one preferred embodiment of the invention,
the housing is sized and adapted for hand-held use.

[0055]In accordance with the present invention there is still further
disclosed yet another test system for use with a buffer to test for the
presence of target molecules of one or more target types in a biological
test sample. According to this embodiment of the invention, the test
system is also for use with a first set of test molecules selected from a
group that includes bio-recognition molecules (BRMs) conjugates of the
BRMs and the target molecules, if present in the test sample. The BRMs
are of one or more BRM types. Each of the BRM types is conjugable with a
respective one of the target types. The conjugates are of one or more
conjugate types, each corresponding to a different one of the BRM types
in conjugation with its aforesaid respective one of the target types. The
test system is additionally for use with an irradiating and detection
device that is capable of delivering electromagnetic frequency (EMF)
radiation for absorption by the test molecules. The test molecules are
such as to emit fluorescence after absorption of the EMF radiation. The
fluorescence of the test molecules includes a distinct fluorescent
spectrum for each one of the conjugate types. The irradiation and
detection device is also capable of monitoring for, and identifying, the
conjugates by the presence of the distinct fluorescent spectrum for each
one of the conjugate types. According to this embodiment of the
invention, the test system includes a microfluidic chip having a chip
substrate portion that is shaped to define one or more elongate sample
channels, and one or more flow focusing channels, therein. The sample
channels are sized to enable passage therethrough of the test molecules.
The flow focusing channels are for operative passage therethrough of the
buffer. The flow focusing channels adjoin the sample channels. The buffer
exits from the flow focusing channels and operatively directs a
single-file stream of the test molecules through at least one of the
sample channels. The microfluidic chip is adapted to operatively receive
the EMF radiation from the irradiating and detection device, at an
irradiation position along the aforesaid at least one of the sample
channels, for absorption by the test molecules in the single-file stream.
The microfluidic chip is adapted to enable the irradiation and detection
device to monitor the single-file stream for the fluorescence emitted by
the test molecules. In this manner, the conjugates are operatively
identifiable by the presence of the distinct fluorescent spectrum for
each one of the conjugate types. As such, the presence of the target
molecules in the test sample is operatively identifiable by the test
system.

[0056]According to an aspect of one preferred embodiment of the invention,
the test system is particularly adapted for use with one or more
biological test samples selected from the group consisting of blood,
urine, sputum, and serum.

[0057]According to an aspect of one preferred embodiment of the invention,
the test system may be used for diagnosis of a disease state selected
from the group consisting of bacterial disease states, viral disease
states, fungal disease states, and vector-induced disease states.

[0058]According to an aspect of one preferred embodiment of the invention,
the test system may be used for diagnosis of one or more infectious
diseases.

[0059]According to an aspect of one preferred embodiment of the invention,
the test system may be used for diagnosis of a condition selected from
the group consisting of HIV, HBV and HCV.

[0060]According to an aspect of one preferred embodiment of the invention,
the test system may be used for simultaneous diagnosis of two or more the
conditions selected from the group consisting of HIV, HBV and HCV.

[0061]In accordance with the present invention there also disclosed a
method of focusing molecules to facilitate a test for the presence of
target molecules of one or more target types in a biological test sample.
The method includes a sample flowing step, a buffer flowing step, and a
sample focusing step after the buffer flowing step. In the sample flowing
step, test molecules are passed through one or more elongate sample
channels formed in a chip substrate portion of a microfluidic chip. In
the buffer flowing step, a buffer is passed through one or more flow
focusing channels formed in the chip substrate portion of the
microfluidic chip. The flow focusing channels adjoin the one or more
elongate sample channels. In the sample focusing step, a single-file
stream of the test molecules is directed through at least one of the
sample channels by passage of the buffer from the flow focusing channels
into the one or more elongate sample channels.

[0062]According to an aspect of one preferred embodiment of the invention,
the method also includes a test molecule-forming step before the sample
flowing step. In the test molecule-forming step, the test molecules are
formed by introducing bio-recognition molecules (BRMs) of one or more BRM
types. Each of the BRM types is conjugable with a respective one of the
target types. As such, the test molecules include conjugates of the BRMs
and the target molecules, if present in the test sample.

[0063]According to an aspect of one preferred embodiment of the invention,
in the test molecule-forming step, the conjugates are less than about 10
micrometers (μm) in size, and preferably less than about 5 μm in
size, and still more preferably, less than about 1 μm in size.

[0064]According to an aspect of one preferred embodiment of the invention,
in the test molecule-forming step, target marker fluorophores are
introduced. The target marker fluorophores are conjugable with one or
more of the target types. As such, the test molecules include conjugates
of the BRMs, the target marker fluorophores, and the target molecules, if
present in the test sample.

[0065]According to an aspect of one preferred embodiment of the invention,
in the sample focusing step, the single-file stream of the test molecules
is directed along a sample path that is in spaced relation from at least
two opposing side channel portions of the aforesaid at least one of the
sample channels.

[0066]According to an aspect of another preferred embodiment of the
invention, in the sample focusing step, the single-file stream of the
test molecules is directed along a sample path that is in spaced relation
from at least top and bottom channel portions of the aforesaid at least
one of the sample channels.

[0067]According to an aspect of one preferred embodiment of the invention,
in the sample focusing step, the buffer flows into the aforesaid at least
one of the sample channels at a buffer flow rate that is higher than a
test flow rate of the test molecules in the single-file stream.

[0068]According to an aspect of one preferred embodiment of the invention,
in the sample focusing step, at least two of the flow focusing channels
adjoin the sample channels, from opposing sides thereof, upstream of said
at least one of the sample channels.

[0069]According to an aspect of one preferred embodiment of the invention,
in the sample focusing step, the two flow focusing channels adjoin the
sample channels at a common intersection portion.

[0070]According to an aspect of one preferred embodiment of the invention,
in the sample focusing step, each of the one or more flow focusing
channels adjoins the sample channels at an adjoining angle of about 90
degrees.

[0071]According to an aspect of another preferred embodiment of the
invention, in the sample focusing step, each of the one or more flow
focusing channels adjoins the sample channels at an adjoining angle of
about 45 degrees.

[0072]According to an aspect of one preferred embodiment of the invention,
in the sample focusing step, passage of the single-file stream of the
test molecules through the aforesaid at least one of the sample channels
is facilitated by electrokinetic flow.

[0073]According to an aspect of one preferred embodiment of the invention,
the method also includes an electrokinetic step before the sample
focusing step. In the electrokinetic step, a first electrical potential
of a first polarity is supplied to the sample channels upstream of the
flow focusing channels. In the electrokinetic step, a second electrical
potential of an opposing second polarity is supplied to the aforesaid at
least one of the sample channels downstream of the flow focusing
channels. In the electrokinetic step, a third electrical potential of the
first polarity is supplied to each one of the flow focusing channels.

[0074]According to an aspect of one preferred embodiment of the invention,
in the electrokinetic step, the third electrical potential is higher than
the first electrical potential.

[0075]According to an aspect of one preferred embodiment of the invention,
in the electrokinetic step, a ratio of the third electrical potential
relative to the first electrical potential is about 1.8:1 (9:5).

[0076]According to an aspect of one preferred embodiment of the invention,
in the test molecule-forming step, the conjugates are of one or more
conjugate types, each corresponding to a different one of the BRM types
in conjugation with its said respective one of the target types. The
method also includes an irradiating step after the sample focusing step,
a fluorescence-detecting step after the irradiating step, and a
conjugate-identifying step after the irradiating step. In the irradiating
step, electromagnetic frequency (EMF) radiation is delivered to the test
molecules in the single-file stream. In the fluorescence-detecting step,
the single-file stream is monitored for fluorescence emitted by the test
molecules. Each of the conjugates, after absorption of the EMF radiation,
emits fluorescence of a distinct fluorescent spectrum for each one of the
conjugate types. In the conjugate-identifying step, the presence of the
target molecules in the test sample is identified by monitoring for the
distinct fluorescent spectrum of each one of the conjugate types.

[0077]According to an aspect of one preferred embodiment of the invention,
in the test-molecule forming step, target marker fluorophores are bound
to respective ones of the target molecules. As such, in the
fluorescence-detecting step, the target marker fluorophores emit a target
part of the distinct fluorescent spectrum for each one of the conjugate
types. The method further includes a BRM-forming step, before the
test-molecule forming step, of tagging a microbead with one or more BRM
fluorophores that are coupled to the microbead. As such, in the
fluorescence-detecting step, the BRM fluorophores emit a BRM part of the
distinct fluorescent spectrum for each one of the conjugate types.

[0078]According to an aspect of one preferred embodiment of the invention,
in the fluorescence-detecting step, fluorescence emitted by the
conjugates is received by at least two avalanche photodetectors (APDs). A
first one of the APDs receives and identifies the presence of the BRM
part, and a second one of the APDs receives and identifies the presence
of the target part, of the fluorescence of the distinct fluorescent
spectrum for said each of the conjugates.

[0079]According to an aspect of one preferred embodiment of the invention,
in the irradiating step, a laser having an operating power of between
about 2 megawatts (mW) and about 50 megawatts (mW) delivers the EMF
radiation to the test molecules in the single-file stream. More
preferably, the operating power is between about 20 megawatts (mW) and
about 25 megawatts (mW).

[0080]According to an aspect of one preferred embodiment of the invention,
in the irradiating step, the EMF radiation has an EMF wavelength of about
488 nm.

[0081]According to an aspect of one preferred embodiment of the invention,
in the fluorescence-detecting step, the fluorescence emitted by the
conjugates is received by a charge-coupled device.

[0082]According to an aspect of one preferred embodiment of the invention,
in the fluorescence-detecting step, the fluorescence emitted by the
conjugates is selectively received by at least one of a charge-coupled
device and one or more avalanche photodetectors (APDs).

[0083]Other and further advantages and features of the invention will be
apparent to those skilled in the art from the following detailed
description thereof, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0084]The invention will now be described in more detail, by way of
example only, with reference to the accompanying drawings, in which like
numbers refer to like elements, wherein:

[0085]FIG. 1A is an illustration of a first conjugate according to the
present invention;

[0086]FIG. 1B is an illustration of a second conjugate according to the
present invention;

[0087]FIG. 1C is an illustration of a third conjugate according to the
present invention;

[0088]FIG. 2A is a perspective view of a microfluidic chip according to
the present invention;

[0089]FIG. 2B is a perspective view of an alternate microfluidic chip
according to the present invention;

[0090]FIG. 2C is a close-up top view of the microfluidic chip intersection
of FIG. 2B;

[0091]FIG. 3A is a schematic of a test system according of the present
invention;

[0092]FIG. 3B is a schematic of the irradiation device of FIG. 3A;

[0093]FIG. 3C is a schematic of the detection device of FIG. 3A;

[0094]FIG. 4A is a perspective view of the system housing;

[0095]FIG. 4B is a perspective view of the system with the housing
removed;

[0096]FIG. 4C is a perspective view of the microchip platform and
irradiation device with the sample hatch closed;

[0097]FIG. 4D is a top view of the microchip platform with the sample
hatch open;

[0098]FIG. 4E is a side view of the lens, motors and microchip platform;

[0099]FIG. 4F is a cross-section schematic of the lens and microfluidic
chip platform along line 4F-4F of FIG. 4E;

[0100]FIG. 4G is a close-up schematic of the indicated section of FIG. 4F
and a cross-section along line 4G-4G of FIG. 2C.

[0101]FIG. 5A is an illustration of fluorescing quantum dots;

[0102]FIG. 5B is an illustration of fluorescing quantum dots;

[0103]FIG. 6A is a spectrum of the quantum dots and bandpass filters used
in the experiments described herein;

[0104]FIG. 6B is a spectrum of the quantum dots of FIG. 5B;

[0105]FIG. 7A is a graph of raw and fitted fluorescent emission wavelength
data for the BRM conjugate of FIG. 1A;

[0106]FIG. 7B is a graph of raw and fitted fluorescent emission wavelength
data for the BRM conjugate of FIG. 1B;

[0107]FIG. 7c is a graph of raw and fitted fluorescent emission wavelength
data for the BRM conjugate of FIG. 1C;

[0108]FIG. 8A is a graph of intensity vs. time measurements for the BRM
conjugates of FIG. 1A;

[0109]FIG. 8B is a graph of intensity vs. time measurements for the BRM
conjugates of FIG. 1B;

[0110]FIG. 8C is a graph of intensity vs. time measurements for the BRM
conjugates of FIG. 1C;

[0111]FIG. 9 is a close-up of the indicated section of FIG. 8A

[0112]FIG. 10A is a graph of intensity vs. concentration measurements for
the BRM conjugates of FIG. 1A;

[0113]FIG. 10B is a graph of intensity vs. concentration measurements for
the BRM conjugates of FIG. 1A;

[0114]FIG. 10C is a graph of intensity vs. concentration measurements for
the BRM conjugates of FIG. 1A;

[0115]FIG. 11A is a histogram of R/Y signal ratios for the BRM conjugates
of FIG. 1A;

[0116]FIG. 11B is a histogram of R/Y signal ratios for the BRM conjugates
of FIG. 1B;

[0117]FIG. 11c is a histogram of R/Y signal ratios for the BRM conjugates
of FIG. 1C;

[0122]Referring now to FIGS. 1A through 4G, there is shown a test system
100 according to a preferred embodiment of the present invention. The
test system 100 is for use with a buffer 50 to test for the presence of
target molecules 46a, 46b, 46c (46a-c) of one or more target types in a
biological test sample 40. The test system 100 preferably includes a
first set of test molecules 102, a microfluidic chip 200, an irradiating
device 300, and a detection device 400. The test system 100 also
preferably includes a housing 500 encasing the irradiating device 300 and
the detection device 400, with housing 500 being sized and adapted for
portable, hand-held, and point-of-care diagnostic use.

[0123]Introduction to the System

[0124]Preferably, the first set of test molecules 102 may include (i)
detection molecules 106a, 106b, 106c (106a-c) and (ii) conjugates 126a,
126b, 126c of the detection molecules 106a-c and the target molecules
46a-c, if present in the test sample 40.

[0125]As best seen in FIGS. 1A through 1C, the detection molecules 106a-c
are of one or more detection molecule types. Each of the detection
molecule types is conjugable with a respective one of the target types.
Each of the detection molecules 106a-c preferably includes a microbead
108 along with one or more--and preferably a plurality
of--bio-recognition molecules 116 (BRMs 116) bound to the surface of the
microbead 108 by a carboxylic acid 118. Each of the BRMs 116 is specific
for one of the target types of the target molecules 46a-c. Each microbead
108 is preferably bound to a plurality of BRMs 116. The plurality of BRMs
116 bound to the microbead 108 may collectively be conjugable with more
than one--but, preferably, are collectively conjugable with only a single
one--of the target types. Each microbead 108 is preferably also tagged
with one or more BRM fluorophores 112a-b that are coupled to the
microbead 108. The BRM fluorophores 112a-b preferably include one or more
quantum dots 112a, 112b of one or more quantum dot types. In some cases,
and as best seen in FIG. 1C, the quantum dots 112a, 112b may be of two or
more quantum dot types--e.g., red quantum dots 112b and yellow quantum
dots 112a. Alternately, the BRM fluorophores 112a-b may include one or
more fluorescent dyes (not shown) of one or more fluorescent dye types.

[0126]As best seen in FIGS. 1A through 1C, the conjugates 126a, 126b, 126c
are of one or more conjugate types each corresponding to a different one
of the detection molecule types in conjugation with its corresponding
target type. The conjugates 126a, 126b, 126c are preferably less than
about 10 micrometers (μm) in size. In some embodiments, the conjugates
126a, 126b, 126c may be less than about 5 μm in size, or even less
than about 1 μm in size. Preferably, and as best seen in FIGS. 1A
through 1C, each of the conjugates 126a, 126b, 126c also includes a
target marker fluorophore 130 bound to a respective one of the target
molecules 46a, 46b, 46c.

[0127]As best seen in FIGS. 2A and 2B, the microfluidic chip 200
preferably includes a chip substrate portion 202 and a glass slide 250
underlying the chip substrate portion 202. The chip substrate portion 202
is preferably fabricated from polydimethylsiloxane (PDMS), which is
shaped to define one or more elongate sample channels 204, and one or
more flow focusing channels 220a, 220b, therein. The flow focusing
channels 220a, 220b are in fluid communication with at least one of the
sample channels 204. Preferably, the chip substrate portion 202 is
additionally shaped to define a sample well 242, two buffer wells 244a,
244b, and a terminal well 246 therein.

[0128]As best seen in FIG. 2C, the sample channels 204 are sized to enable
passage therethrough of the test molecules 102. As best seen in FIGS. 2A
to 2C, the sample channels 204 include a sample supply channel 206, and a
sample focused channel 208 in fluid communication with the sample supply
channel 206. The sample well 242 is adjacent to a sample starting point
212 of the sample supply channel 206--i.e., upstream (i.e., in a
direction generally opposed to arrow "A") of the flow focusing channels
220a, 220b. The sample focused channel 208 is downstream (in the
direction indicated generally by arrow "A") of the flow focusing channels
220a, 220b. The terminal well 246 is adjacent to an end point 216 of the
sample focused channel 208--i.e., downstream (in the direction indicated
generally by arrow "A") of the flow focusing channels 220a, 220b. As best
seen in FIG. 4G, the sample focused channel 208 is defined by one or more
elongate channel walls 284 of the chip substrate portion 202. The channel
walls 284 include a top channel portion 282a and two opposing side
channel portions 282c. As best seen in FIG. 4G, the glass slide 250
defines a bottom channel portion 282b of the sample focused channel 208.

[0129]The flow focusing channels 220a, 220b are for operative passage
therethrough of the buffer 50. As best seen in FIG. 2C, there are
preferably two flow focusing channels 220a, 220b, which adjoin the sample
channels 204 upstream (i.e., in a direction generally opposed to arrow
"A"), and from opposing sides 282c,282c, of the sample focused channel
208. As shown in FIG. 2A, each buffer well 244a, 244b is adjacent to a
buffer starting point 214a, 214b of a respective one of the flow focusing
channels 220a, 220b. Each of the flow focusing channels 220a, 220b may
adjoin the sample channels 204 at an adjoining angle (as indicated
generally by arrow "E" in FIGS. 2A and 2C) of about 90 degrees (as shown
in FIG. 2A), about 45 degrees (as best seen in FIG. 2C), or another
potentially advantageous adjoining angle "E". As shown in FIGS. 2A and
2B, the two flow focusing channels 220a, 220b preferably adjoin the
sample channels 204 at a common intersection portion 230.

[0130]As shown in FIG. 2C, the buffer 50 exits from the flow focusing
channels 220a, 220b and operatively directs a single-file stream 140 of
the test molecules 102 through at least one of the sample channels (i.e.,
the sample focused channel 208)--preferably by less than about 10
micrometers (μm) downstream "A" of the common intersection portion
230. The buffer 50 also operatively flows through the sample focused
channel 208. As indicated generally by the relative lengths of arrows
"B", "D1" and "D2" in FIG. 2C, a buffer flow rate (as indicated generally
by arrows "D1", "D2") of the buffer 50 is typically higher than a test
flow rate (as indicated generally by arrow "B") of the test molecules 102
in the single-file stream 140. The single-file stream 140 is directed
along a sample path (as indicated generally by arrow "B") that is
preferably in spaced relation from the opposing side channel portions
282c,282c (as best seen in FIG. 2C), and from the bottom channel portion
282b and the top channel portion 282a (as best seen in FIG. 4G).

[0131]Passage of the test molecules 102 through the sample focused channel
208 is preferably facilitated by electrokinetic flow. Accordingly, and as
best seen in FIG. 4D, the test system 100 preferably also includes a
sample well electrode 262, two buffer well electrodes 264a, 264b, and a
terminal well electrode 266. The positioning of the electrodes 262, 264a,
264b and 266 may be best appreciated from a consideration of FIGS. 4D and
4E. The sample well electrode 262 is operatively positioned in the sample
well 242. Each buffer well electrode 264a, 264b is operatively positioned
in one of the buffer wells 244a, 244b. The terminal well electrode 266 is
operatively positioned in the terminal well 246.

[0132]The sample well electrode 262 is operatively supplied with a first
electrical potential of a first polarity. The terminal well electrode 266
is operatively supplied with a second electrical potential of an opposing
second polarity. Each buffer well electrode 264a, 264b is operatively
supplied with a third electrical potential of the first polarity.
Preferably, the third electrical potential is higher than the first
electrical potential, with a ratio of the third electrical potential
relative to the first electrical potential being about 1.8:1 (9:5).

[0133]Preferably, the test flow rate "B" of the test molecules 102 in the
single-file stream 140 is at least about thirty (30) test molecules 102
per minute. More preferably, the test flow rate "B" may be at least about
sixty (60) test molecules 102 per minute. Preferably, even higher test
flow rate "B"s--e.g., about five hundred (500) test molecules 102 per
minute--may afford even more advantageous utility.

[0134]As best seen in FIGS. 3A and 3B, the irradiating device 300
operatively delivers electromagnetic frequency (EMF) radiation 302, at an
irradiation position 210 (best seen in FIGS. 2A and 2B) along the sample
focused channel 208, for absorption by the test molecules 102 in the
single-file stream 140. The irradiating device 300 may include an LED 312
(as best seen in FIGS. 4E and 4F) and/or a laser 310 (as best seen in
FIGS. 3A and 3B) to operatively emit the EMF radiation 302. Preferably,
the laser 310 has an operating power of between about 2 megawatts (mW)
and about 50 mW, and more preferably, between about 20 mW and about 25
mW. Preferably, the EMF radiation 302 may have an EMF wavelength of about
488 nm.

[0135]As best seen in FIGS. 3A to 3C, the test molecules 102 emit
fluorescence 304 after absorption of the EMF radiation 302. The
fluorescence 304 of the test molecules 102 includes a distinct
fluorescent spectrum 726a, 726b, 726c (as best seen in FIGS. 7A to 7C)
for each one of the conjugate types. Preferably, and as best seen in
FIGS. 6A to 7C, after absorption of the EMF radiation 302, the BRM
fluorophores 112a-b--whether quantum dots 112a, 112b or fluorescent dyes
(not shown)--emit at least a BRM part 604a, 604b, 604c, and the target
marker fluorophore 130 emits a target part 604d, of the fluorescence 304
of the distinct fluorescent spectrum 726a, 726b, 726c after absorption of
the EMF radiation 302. (The target part 604d may have a lower intensity
than the BRM part 604a, 604b, 604c, of the fluorescence 304 of the
distinct fluorescent spectrum 726a, 726b, 726c for each of the conjugates
126a, 126b, 126c.) The BRM fluorophores 112a, 112b and the target marker
fluorophore 130 together emit the fluorescence 304 of the distinct
fluorescent spectrum 726a, 726b, 726c after absorption of the EMF
radiation 302.

[0136]Though not shown in the figures, the test system 100 may alternately
include a fiber optic cable delivering the fluorescence 304 to the
detection device 400 from substantially adjacent to the irradiation
position 210 (i.e., along the sample focused channel 208).

[0137]As may be best appreciated from a consideration of FIGS. 3A, 3C, 6A
and 7A-9, the detection device 400 monitors the single-file stream 140
for the fluorescence 304 emitted by the test molecules 102. Generally
speaking, the detection device 400 identifies the presence of the
conjugates 126a, 126b, 126c in the first set of test molecules 102 by
monitoring for the distinct fluorescent spectrum 726a, 726b, 726c (as
best seen in FIGS. 7A to 7C) of each one of the conjugate types.

[0138]As best seen in FIG. 3C, the detection device 400 may preferably
include avalanche photodetectors 410a, 410b, 410c, 410d (APDs 410a-d), a
charge-coupled device 420, and/or one or more trip sensors (not shown),
so as to monitor the single-file stream 140 for the fluorescence 304
emitted by the test molecules 102. Preferably, the detection device 400
includes two, three, four, or more APDs 410a-d monitoring the single-file
stream 140 for the fluorescence 304 emitted by the test molecules 102.
Each of the APDs 410a-d is preferably adapted to receive and identify the
presence of a different range of wavelengths--e.g., blue (not shown),
green 602d, yellow 602a, orange 602c, or red 602b ranges of wavelengths.

[0139]In some embodiments of the invention, a first one of the APDs 410a-d
may preferably be adapted to receive and identify the presence of the BRM
part 604a, 604b, 604c, with a second one of the APDs 410a-d being adapted
to receive and identify the presence of the target part 604d, of the
fluorescence 304 of the distinct fluorescent spectrum 726a, 726b, 726c
for each of the conjugates 126a, 126b, 126c. In such embodiments, the
second one of the APDs 410a-d may preferably have a greater sensitivity
than the first one of the APDs 410a-d. Where the detection device 400
additionally includes a charge-coupled device 420, it may also include a
switch means 464 (as best seen in FIG. 3C) for switching between
monitoring the single-file stream 140 with either the APDs 410a-d or the
charge-coupled device 420.

[0140]As aforesaid, and though not shown in the figures, the detection
device 400 may include a series of one or more trip sensors. Each such
trip sensor may preferably generate a digital signal corresponding to an
intensity 802a, 802b, 802c (as best seen in FIGS. 8A-9) of the
fluorescence 304, but only when the intensity of the fluorescence 304 is
in excess of a minimum trip intensity. Each trip sensor in the series may
preferably be provided with a different pre-determined minimum trip
intensity. The series of trip sensors may preferably be arranged in
ascending or descending order of minimum trip intensities.

[0141]In this manner, the test system 100 identifies the presence of the
target molecules 46a, 46b, 46c in the test sample 40. Preferably, the
test system 100 is particularly suited for use with blood, urine, sputum,
and serum test samples. It is intended to be used for diagnosis of
infectious diseases, and/or of bacterial disease states, viral disease
states, fungal disease states, and/or vector-induced disease states. In
particular, the test system 100 may be particularly useful in
simultaneously diagnosing whether an organism is infected with HIV, HBV
or HCV.

[0142]At this stage, it may be worthwhile to specifically note that, in
some embodiments falling within the scope of the invention, the test
system 100 may be provided without (though preferably still for use with)
one or more of its aforementioned component parts. That is, and for
example, the test system 100 may be provided without the test molecules
102, though it might still be intended for use with same. Similarly, the
test system 100 may be provided without one or more of the microfluidic
chip 200, the irradiating device 300, and the detection device
400--though, of course, it might still be intended for use with same. For
example, where the test system 100 is provided with neither the
irradiating device 300 nor the detection device 400, it may be intended
for use with a combined irradiating and detection device 300, 400.

[0143]Introduction to the Method

[0144]In accordance with the present invention there also disclosed a
method, inter alia, of focusing molecules to facilitate a test for the
presence of target molecules 46a, 46b, 46c of one or more target types in
a biological test sample 40. The method preferably includes:

[0154]In the detection molecule forming step, a microbead 108 is tagged
with one or more BRM fluorophores 112a, 112b that are coupled to the
microbead 108. In the conjugate-forming step, the conjugates 126a-c are
preferably formed by introducing target marker fluorophores 130 and
detection molecules 106a-c (of one or more detection molecule types) into
the biological test sample. Each of the detection molecule types is
conjugable with a respective one of the target types, and the target
marker fluorophores 130 is preferably conjugable/bindable with one or
more (and/or all) of the target types. As such, if the target molecules
46a, 46b, 46c are present in the test sample, the test molecules 102 may
preferably include conjugates 126a, 126b, 126c of the detection molecules
106a-c, the target marker fluorophores 130, and the target molecules 46a,
46b, 46c. Preferably, the conjugates 126a, 126b, 126c formed in the
conjugate-forming step are less than about 10 micrometers (μm) in
size. In some embodiments, the conjugates 126a, 126b, 126c formed may be
less than about 5 μm in size, or even less than about 1 μm in size.
The conjugates 126a, 126b, 126c so formed are of one or more conjugate
types, each corresponding to a different one of the detection molecule
types in conjugation with the corresponding target type.

[0155]In the electrokinetic step: (i) a first electrical potential of a
first polarity is supplied to the sample supply channel 206, i.e.,
upstream (i.e., in a direction generally opposed to arrow "A") of the
flow focusing channels 220a, 220b; (ii) a second electrical potential of
an opposing second polarity is supplied to the sample focused channel
208, i.e., downstream "A" of the flow focusing channels 220a, 220b; and
(iii) a third electrical potential of the first polarity is supplied to
each of the flow focusing channels 220a, 220b. The third electrical
potential is preferably higher than the first electrical potential. A
ratio of the third electrical potential relative to the first electrical
potential is preferably about 1.8:1 (i.e., about 9:5). In the sample
flowing step, the test molecules 102 are passed through the sample supply
channel 206. In the buffer flowing step, the buffer 50 is passed through
the flow focusing channels 220a, 220b, adjoining the sample channels 204.

[0156]In the sample focusing step, a single-file stream 140 of the test
molecules 102 is directed through the sample focused channel 208 by
passage of the buffer 50 from two flow focusing channels 220a, 220b into
the sample focused channel 208 via an adjoining common intersection
portion 230. The single-file stream 140 is directed along a sample path
"B" that is in spaced relation from the opposing side channel portions
282c,282c, from the top channel portion 282a, and from the bottom channel
portion 282b of the sample focused channel 208. Typically, the buffer 50
flows into the sample focused channel 208 at a buffer flow rate "D1",
"D2" that is higher than a test flow rate "B" of the test molecules 102
in the single-file stream 140. In the sample focusing step, the buffer 50
may flow into the sampled focused channel from an adjoining angle "E" of
about 90 degrees (as shown in FIG. 2A), about 45 degrees (as best seen in
FIG. 2C), or from another potentially advantageous adjoining angle "E".
Preferably, in the sample focusing step, passage of the single-file
stream 140 of the test molecules 102 through the sample focused channel
208 is facilitated by the electrokinetic step.

[0157]In the irradiating step, electromagnetic frequency (EMF) radiation
302 is delivered to the test molecules 102 in the single-file stream 140,
preferably by a laser 310 having an operating power of between about 2 mW
and about 50 mW. More preferably, the operating power may be between
about 20 mW and about 25 mW. In one preferred embodiment, the EMF
radiation 302 has an EMF wavelength of about 488 nm.

[0158]After absorption of the EMF radiation 302, each of the conjugates
126a, 126b, 126c (i.e., of each conjugate type) emits fluorescence 304 of
a distinct fluorescent spectrum 726a, 726b, 726c. The target marker
fluorophores 130 emit a target part 604d, and the BRM fluorophores 112a,
112b emit a BRM part 604a, 604b, 604c, of the distinct fluorescent
spectrum 726a, 726b, 726c for each conjugate type.

[0159]In the fluorescence-detecting step, the single-file stream 140 is
monitored for fluorescence 304 emitted by the test molecules 102. The
fluorescence 304 emitted by the conjugates 126a, 126b, 126c is preferably
received by two or more APDs 410a-d--with first and second APDs 410a-d
receiving and identifying the BRM part 604a, 604b, 604c and the target
part 604d, respectively, of the fluorescence 304 of the distinct
fluorescent spectrum 726a, 726b, 726c for each of the conjugates 126a,
126b, 126c. Alternately, the fluorescence 304 emitted by the conjugates
126a, 126b, 126c may be received by a charge-coupled device 420. Still
further, in the fluorescence-detecting step, the fluorescence 304 emitted
by the conjugates 126a, 126b, 126c may be selectively received by the
APDs 410a-d, the charge-coupled device 420, or both.

[0160]Finally, in the conjugate-identifying step, the presence of the
target molecules 46a, 46b, 46c in the test sample 40 is identified when
the distinct fluorescent spectra 726a, 726b, 726c of the conjugate types
is detected.

[0161]The System in Greater Detail

[0162]The test system 100 according to the invention will now be discussed
in considerably greater detail.

[0163]The test system 100 is designed to test biological test samples
(i.e. blood, sputum, serum, urine, etc.) for various conditions and
infectious diseases in the host who provided the sample. Infectious
diseases tested can include, but are not limited to, bacterial disease
states, viral diseases states, fungal disease states, vector-induced
diseases states, and combinations thereof. Testing is performed by
combining detection molecules 106a-c with the biological sample to form a
test sample 40.

[0164]The test molecules 102 may preferably include conjugates 126a-c as
illustrated in FIGS. 1A, 1B and 1C. Each of the conjugates 126a-c
preferably includes a detection molecule 106a-c. The detection molecule
106a-c preferably includes a polymer microbead 108, with embedded BRM
fluorophores, such as quantum dots 112a-b creating a unique spectral
pattern or "barcode" associated with each detection molecule 106a-c. The
detection molecule 106a-c preferably further includes a BRM 116 bound to
the surface of the microbead 108 by a carboxylic acid 118. The target
molecule 46a-c with its target marker fluorophore 130 is thus bound to
the exterior of the microbead 108 through BRM 116 to form the conjugate
126a-c.

[0165]More specifically, with reference to FIG. 1A, the microbead 108 has
embedded BRM fluorophores shown as yellow quantum dots 112a. FIGS. 1B and
1C show other various combinations of red (112b), and red (112b) and
yellow (112a). As also shown for reference in FIGS. 5A and 5B, orange
(112c), green (112d) and blue (112e) quantum dots can also be used.
Alternative fluorophores, such as fluorescent dyes, can be used in place
of quantum dots. Each BRM fluorophore produces a distinct fluorescent
spectrum, such as shown in FIG. 6A--e.g., distinct fluorescent spectrum
604b for red quantum dots. The target type 46a is conjugated to detection
molecule 106a to form, together with the target marker fluorophore 130,
the conjugate 126a. Similarly, in FIGS. 1B and 1C, target types 46b and
46c are conjugated to detection molecules 106b and 106c to form, together
with the target marker fluorophores 130, conjugates 126b and 126c,
respectively.

[0166]Assembly

[0167]FIG. 3A shows a schematic of an embodiment the present inventive
system 100. The test system 100 is generally comprised of a microfluidic
chip 200, and irradiating device 300 and a detection device 400.

[0168]The microfluidic chip 200, as best shown in FIGS. 2A and 2B,
comprises a chip substrate portion 202 mounted on a glass slide 250. The
chip substrate is comprised of a number of wells and connecting channels.
As shown in FIG. 2B, the exemplary chip 200 has four wells: a sample well
242, two buffer wells 244a and 244b, and a terminal well 246. The sample
well 242 is connected at sample starting point 212 to a sample channel
204, which has two parts, a sample supply channel 206 and a sample
focused channel 208. Similarly, the buffer wells 244a and 244b are
respectively connected at buffer starting points 214a and 214b to flow
focusing channels 220a and 220b. The sample supply channel 206 joins the
flow focusing channels 220b and 220b at a common intersection 230, with
the resulting focused buffer/sample flow entering sample focused channel
208 and terminating at end point 216 into terminal well 246. Along sample
focused channel 208 is an irradiating position 210 for aiming the
irradiating device 300.

[0169]Two variants of the flow focusing channels 220a and 220b are shown
in FIGS. 2A and 2B respectively. In FIG. 2A, the flow focusing channels
220a and 220b enter the intersection 230 at substantially a 90-degree
angle to the sample supply channel 206 and sample focused channel 208. In
FIG. 2B, the flow focusing channels 220a and 220b enter the intersection
230 at substantially a 45-degree angle to the sample supply channel 206.
The geometry of the channels 204 and the angle at the intersection 230 is
preferably between a 30-degree and a 90-degree angle of intersection,
however, the exact angle is best determined by empirical measurement
based on the characteristics of the test sample 40 and buffer 50, as well
as the desired flow rate.

[0170]Based on FIG. 2B, FIG. 2C provides a close-up cross-sectional
schematic of the intersection 230 and the flow patterns of the sample 40
and buffer 50. The general direction of flow towards the terminal well
246 is indicated by arrow A, representing a downstream direction. Sample
flow along sample supply channel 206 is indicated by arrow B,
representing a sample path. The buffer flow along flow focusing channels
220a and 220b is indicated by arrows C2 and C1, respectively. Buffer flow
in the sample focused channel 208 is indicated by arrows D2 and D1. The
angle of incidence between the flow focusing channels 220a and 220b and
the sample supply channel 206 is shown as E.

[0171]As the buffer 50 exits the flow focusing channels 220a and 220b into
the intersection, the force of the flowing buffer 50 causes the flowing
sample 40 from the sample supply channel 206 to narrow and force the test
molecules 102 into a single file stream 140. FIG. 4G shows a
cross-section of sample focused channel 208, illustrating that the flow
focusing effect of buffer 50 on sample 40 function in the both
directions, subject to constraint by the channel walls 284. Note that
sample 40 extends to the bottom channel portion 282b defined by the glass
slide 250, but narrowly, to prevent adhesion of the test molecules 102 to
the bottom channel portion 282b. The buffer 50 covers opposing side
channel portions 282c, while both the buffer 50 and sample 40 cover the
top channel portion 282a.

[0172]The microfluidic chip 200 is mounted on a platform 270 as best shown
in detail in FIGS. 4E and 4F. The electrokinetic driving force for the
sample 40 and buffer 50 is provided by electrodes that are inserted into
each of the wells. Thus, for the chip 200 shown, there are a sample well
electrode 262, two buffer well electrodes 264a and 264b, and a terminal
well electrode 266. A voltage differential is applied via the electrodes
to produce the electrokinetic driving force which carries the sample 40
and buffer 50 along their respective channels.

[0173]The microfluidic chip 200 is manufactured according to known
methods. One such method uses a polydimethylsiloxane (PDMS) microfluidic
chip. The PDMS microfluidic chips are preferably fabricated using
conventional soft lithography microfabrication techniques. Photomasks of
the desired microchannel pattern are prepared and printed on a
transparency. A master is fabricated on Si wafers coated with a layer of
photoresist and prebaked. Each wafer then has the photomask laid on top
of the photoresist, ink surface down, and is exposed to UV light for a
brief duration. Following standard postbaking procedures, the wafers are
immersed in nanodeveloper to dissolve away any photoresist not exposed to
the UV light. The masters are then washed with isopropanol and dried with
compressed N2 gas.

[0174]The polydimethylsiloxane (PDMS) is generally supplied as prepolymer
kits in two parts; part A is the prepolymer and part B contains a
cross-linker. The masters are placed in pyrex Petri dishes and mixed
prepolymer was poured on top of each. The samples are then placed under
vacuum to degas (remove bubbles from) the prepolymer. An incubation
period follows in an oven. Once removed from the oven, the cured PDMS
slabs are peeled off the masters and excess polymer around the outside of
the microchannel pattern is removed. A single master holds patterns for
two polymer microchannels. The surfaces of the PDMS substrates and glass
coverslips is then cleaned using scotch tape.

[0175]Plasma oxygen pretreatment of the PDMS channels can then be used to
make the walls hydrophilic. Both the PDMS substrates 202 and glass slides
250 are loaded into the chamber of a plasma cleaner and exposed to oxygen
plasma. Immediately after, the surfaces of the PDMS 202 and slides 250
are brought into contact to irreversibly seal the two substrates
together. Double distilled water is dispensed into the microchannels 204
to keep the channel surfaces hydrophilic. Finally, small pieces of glass
are placed on top of the channel wells 242, 244a, 244b and 246 to keep
the water from evaporating, enabling long term storage of the chips 200.

[0176]The irradiating device 300, shown in greater detail in FIG. 3B,
emits a stream of EMF (electromagnetic frequency) radiation 302 which is
directed onto the irradiating position 210 to irradiate the test
molecules 102 flowing in the sample focused channel 208 as discussed
above. As shown, laser 310 is used to produce the EMF radiation, however,
alternative sources, such as an LED can also be used. The LED can be
positioned in the same location as laser 310, or in the position shown by
LED 312.

[0177]As shown in FIG. 4F, the emitted EMF radiation 302 is directed via a
mirror 320 through an objective lens 330 to focus the beam onto the
irradiating position 210. The focal length of the lens can be adjusted
along the x-, y-, and z-axis (as also shown in FIGS. 4C and 4D) by
movement of the chip platform 270 using motors 280a and 280b to align the
radiation 302 with the irradiating position 210. Alignment can be
performed automatically or manually. Automatic alignment is performed by
activating LED 312 and measuring the intensity of the signal, and
adjusting the platform in the x- and z-axis for proper alignment with the
sample focused channel 208 and irradiating position 210. Alternatively,
the y-axis adjustment can be conducted manually to optimize the signal
results. Manual alignment is controlled by the user through of adjustment
knob 506, the input interface 504, or a combination thereof.

[0178]The alignment and focal adjustment of the lens 330 is more clearly
shown in FIG. 4G, with the z-axis focal distance, Fz, between the
lens 330 and the test molecules 102 being adjusted to ensure the test
molecules 102 are sufficiently excited by the EMF radiation 302.
Similarly, the x-axis focal distance, Fx, is adjusted to account for
the size (width) of the test molecules 102.

[0179]The detection device 400, shown in greater detail in FIG. 3C,
generally comprises a collection of mirrors and filters to direct the
emitted fluorescence 304 from the irradiated test molecules 102 into
fluorescence detection devices. As shown in FIG. 3C, incoming
fluorescence 304 is directed along a detection channel 450 by a primary
mirror 462. The fluorescence signal is then split by a beam splitter
mirror 464. One portion of the split fluorescence signal is directed
along an intensity channel 422 to a Charge-Coupled Device ("CCD") 420
which determines the overall intensity of the signal.

[0180]The other portion of split fluorescence signal is directed along a
detection channel 450 where it passes through a series of bandpass
filters 440a-d. Each filter 440a-d covers a specific wavelength
corresponding to the fluorescence signals 304 emitted by each of the BRM
fluorophores 112a-b and target marker fluorophores 130 in the test
molecules 102. The filtered signals 442a-d are each directed along
detection channels 452a-d to APDs (Avalanche PhotoDetectors) 410a-d that
convert the fluorescence signal into an electrical signal which is then
output to a signal processor 490 for analysis.

[0181]Taking one signal as an example, a green wavelength bandpass filter
440a is used to divert a filtered portion 442a of the fluorescence signal
304 into detection channel 452a. This filtered signal 442a impacts APD
410a and the result is a green wavelength output signal for analysis. A
similar process takes place using yellow bandpass filter 440b, orange
bandpass filter 440c and red bandpass filter 440d, with corresponding
APDs 410b-d producing output signals for the yellow, orange and red
wavelengths. The combined signals collectively produce a spectrum, which
is interpreted to determine the identity of the test molecules 102 that
have fluoresced.

[0182]As the target fluorophore 130 is generally of lower, often
substantially lower, intensity than the BRM fluorophores 112a-b, it can
be advantageous to have the APD responsible for generating the target
fluorophore spectrum to operate at a higher sensitivity than the APDs
responsible for generating the BRM conjugate spectra. In the schematic
shown, APD 410a is responsible for generating the spectrum of target
fluorophore 130, and operates with a greater sensitivity than APDs 410b-d
used for the BRM fluorophores 112a-b. As shown, APD 410a is of a type
that uses a heat sink 470 to cool the APD, providing greater sensitivity
over uncooled APDs 410b-d. Alternately, or in addition to heat sink 470,
a temperature control system 472 can be implemented to maintain APD 410a
at a constant temperature below ambient.

[0183]The overall system is encased in housing 500, which includes sample
access port 502 for insertion and removal of sample-loaded microfluidic
chips 200, and display 504, which is preferably a touch-screen device to
enable dual function as a data-entry device. The housing also includes
knobs 506 used to perform manual alignment an adjustment of the position
of the chip 200.

[0184]Operation

[0185]In use, a biological test sample (blood, sputum, serum, urine, etc.)
is prepared for insertion into the sample well 242 of the microfluidic
chip 200. The biological test sample is combined with a first set of
detection molecules to form a test sample 40 which is tested by the
system for the presence of target molecules 46 of one or more target
types, as determined by the nature of the test. Detection molecules 106
of one or more detection molecule types are individually conjugable with
one of the target molecules. Different conjugates of the detection
molecules and the target molecules correspond to different detection
molecules and target molecules.

[0186]A second set of molecules may also be present in the sample, such as
unconjugated sample molecules, and the molecules in the second set might
travel interspersed in the sample flow with the test molecules (and
detection molecules) in the first set. The second set of molecules can be
used for multiplexed tests of separate detection molecules, or for system
tests such as calibration and error-checking, or ignored as non-relevant.

[0187]The microfluidic chip 200 is then inserted into the test system 100
through the sample access port 502 in the housing 500. Alignment of the
lens 330 and irradiation position 210 is then performed as discussed
above. Operation parameters are input through the display 504 and the
necessary electrical potential is applied through electrodes 262, 264a,
264b and 266 to commence flowing of the sample 40 and buffer 50. A first
electrical potential is applied to sample well electrode 262, a second
electrical potential to buffer well electrodes 264a and 264b, and a third
electrical potential to terminal well electrode 266.

[0188]The sample 40 and buffer 50 then flow through the sample supply
channel 206 and through flow focusing channels 220a and 220b,
respectively, as shown in FIG. 2C. The channels 206, 220a and 220b meet
at a common intersection 230 and the flow of the sample 40 is focused
into a single-file stream 140 of test molecules 102.

[0189]The test molecules 102 are then irradiated at an irradiating
position 210 by an irradiation device 300. Preferably, an EMF radiation
device of a fixed wavelength, such as a laser 310 or LED, is used. The
test molecules then emit fluorescence, according to their detection
molecule type and/or conjugate type, each having a distinct fluorescent
spectrum.

[0190]The fluorescence is then detected by a photodetection device, such
as an APD or CCD as discussed above, and the resulting signals can be
output to a signal processor to identify the conjugate types in the test
sample.

[0191]Experimental Results

[0192]In the example described herein, three pathogens (hepatitis B
virus--HBV, hepatitis C virus--HCV, and human immunodeficiency
virus--HIV, as illustrated in FIGS. 1A, 1B and 1C, respectively) were
selected to demonstrate the utility of this integrated device for
infectious disease diagnostics. These three pathogens are all blood-borne
viruses, using similar routes of transmission and are among the most
prevalent diseases in the world with a significant impact on overall ID
morbidity and mortality. For example, HIV infects 40 million, HBV infects
400 million, and HCV infects 170 million people worldwide with an
estimated morbidity rate of 39.5 million for HIV, 350 million chronic HBV
infections, and 130 million chronic HCV infections. The majority of these
cases are located in the developing world. Current diagnostic schemes
require three separate tests and relatively large amounts of blood for
pathogen detection. These requirements create a significant negative
impact on the cost of analysis and speed of analysis. For the developing
world, the implementation of a universal diagnostic device has the
potential to save many lives.

[0193]Quantum Dot Synthesis

[0194]CdSe core ZnS capped quantum dots ("Qdots") were synthesized using
prior art organometallic methods. Briefly, 12-20 g of
tri-noctylphosphineoxide (TOPO, 98% pure, Sigma Aldrich, St. Louis, Mo.)
was heated in a three neck flask to 150° C. under Ar gas. 160
μL of dimethylcadmium (97%, Strem Chemicals, Newburyport, Mass.) was
injected and mixed in with the heated TOPO for ˜15 minutes. After
three purges under vacuum, the contents of the three neck flask were
heated to 350° C. A 2 molar precursor solution of selenium (Se
powder, 99.5%, Sigma Aldrich) and tri-n-octylphosphine (TOP, Sigma
Aldrich) was then injected into the three neck and the temperature
quickly lowered to 300° C. Cd:Se ratios in the ranges of 1.5:1 to
2.5:1 were used. Qdot emission was tracked during the growth phase by
measuring the emission profile of aliquots of the solution in the three
neck flask using a fluorimeter (FluoroMax-3, Jobin Yvon Horiba, Edison,
N.J.). Once the desired peak emission wavelength had been reached,
capping precursor solution consisting of diethyl zinc (Sigma Aldrich),
hexamethyldisilathiane (TMS2(S), Sigma Aldrich) and TOP was injected into
the three neck drop wise at a rate of ˜1 mL/min.

[0195]Following Qdot capping, the three neck temperature was lowered to
<60° C. and chloroform was added. Several washes with methanol
and chloroform (in a 2:1 ratio) were used to precipitate out
nanoparticles from unreacted precursors. The final TOPO coated Qdots were
stored in chloroform until use.

[0196]Quantum Dot Barcode Synthesis

[0197]Qdot barcodes or BRM parts (hereinafter "QdotBs") were prepared
using known methods (M. Han, X. Gao, J. Z. Su, S. Nie, Nat. Biotech., 19,
631 (2001); X. Gao, S. Nie, Anal. Chem., 76, 2406 (2004)). Briefly, 5
μm diameter polystyrene microbeads (Bangs Laboratories, Fishers, Ind.)
with carboxylic acid functional groups on the surface were swollen in
propanol and TOPO-coated Qdots in chloroform were added (roughly
1.5×107 beads in 1 mL of propanol and <100 μL of Qdots in
chloroform). Owing to hydrophobic-hydrophobic interaction, the Qdots
diffused into the microbead interior. The incubation lasted 1 hour for
QdotB1 (570 nm emitting Qdots only) and QdotB2 (615 nm emitting Qdots
only) samples, while for QdotB3, the incubation was split into two steps
with 570 nm emitting Qdots added for the whole hour incubation and 615 nm
emitting Qdots added only for the second half hour. The samples were
washed several times (between 7-10) with propanol and stored in a fridge
at 4° C. until used for an assay. The interval of time between
bead preparation and the start of an assay did not exceed 12 hrs.

[0200]QdotBs prepared in propanol were vortexed, sonicated for 10 seconds
and then run through a 5 mL filter (Falcon, VWR). Samples initially
suspended in 1 mL of propanol at a concentration of 1.5×107
beads/mL were split into 250 μL aliquots and centrifuged at 8000 rpm
for 3 minutes. The supernatant was aspirated and the QdotBs were
resuspended in 100 μL of 0.1 M MES buffer (pH 5.5). Two more washes of
the beads with MES buffer were completed and the samples were then
resuspended in 90 μL of MES buffer. A stock solution of 0.0092 g
N-dimethylaminopropyl-N'-ethylcarbodiimide (EDC, Sigma Aldrich) in 1 mL
MES buffer was prepared and 5 μL were added to each sample. Samples
were then incubated on a vortex, inducing a light shake, for 15 minutes.

[0201]Following the EDC incubation, samples were centrifuged at 9000 rpm
for 3 minutes and aspirated. A wash with 100 μL of MES buffer
followed, with centrifugation again at 9000 rpm. An antigen solution was
prepared at a concentration of 34.4 μg/mL in carbonate-bicarbonate
buffer (pH 9.4). The antigens used were hepatitis B surface antigen
(HBsAg, Advanced Immunochemical, Long Beach, Calif.), non-structural
protein 4 (NSP4, Advanced Immunochemical) and glycoprotein 41 (gp41,
Advanced Immunochemical) for hepatitis B virus (HBV), hepatitis C virus
(HCV) and human immunodeficiency virus (HIV), respectively. The diluted
antigen stock solutions were added to the samples to a final volume of
100 μL followed by a 15 minute incubation on a vortex.

[0202]After incubation with antigen solution, samples were centrifuged at
6500 rpm for 3 minutes, then aspirated. The QdotBs were then resuspended
in 100 μL of quenching buffer (50 mM Glycine and 0.1% Tween) and
incubated for another 15 minutes on a vortex. Following this incubation,
samples were centrifuged at 5500 rpm for 3 minutes, aspirated and
resuspended in 100 μL of 3% milk in phosphate buffer saline (PBS). A
subsequent incubation on a vortex for 30 minutes served to block the
QdotBs with milk proteins. Finally, the QdotBs were washed one more time
with TRIS wash buffer (pH 8.0), using centrifugation at 5000 rpm. This
sample could be stored dry over night if necessary.

[0203]Stock solutions of target antibody solutions were then prepared in
human serum. For HBV, clone X12 anti-HBsAg was used (Advanced
Immunochemical), for HCV clone 8A1 anti-HCV NS-4 was used (Biodesign
International, Saco, Me.) and for HIV, clone 5A1 anti-HIV-1 gp41 was used
(Biodesign International). The Antigen-coated QdotBs were resuspended in
spiked human serum samples to a final volume of 100 μL. They were then
incubated on a vortex for 15 minutes, followed by two washes using TRIS
wash buffer, centrifuging samples at 5000 rpm.

[0204]A stock solution of AlexaFluor-488 dye conjugated goat anti-mouse
IgG antibodies (Invitrogen, Burlington, ON) was diluted 1:300 in TRIS
wash buffer. 100 μL of this solution was used to resuspend each
sample. Samples were covered in tinfoil (to prevent organic dye
photobleaching) and placed on a vortex for 15 minutes. Two final washes
of the QdotB-complexes using 100 μL of TRIS wash buffer were completed
before resuspending the samples in 500 μL of TRIS wash buffer for
short term storage.

[0205]Assay Preparation

[0206]For the multiplexed assays, antigen-coated QdotBs were prepared as
described above. All experiments used approximately the same number of
total beads during antibody capture. If there were two types of antigen
QdotBs being used, then half the microbeads in a sample corresponded to
one code, while the rest corresponded to the other. The same method was
used for samples that used three different QdotBs.

[0207]For the incubation of QdotBs with target antibody-spiked human
serum, a total volume of 100 μL, was always used. Therefore, if a
sample was incubated with two different targeting antibodies, then 50
μL of each spiked serum solution were added. Similarly, if three
different targeting antibodies were to be incubated, 33 μL of each
solution were added.

[0208]FIG. 12 lists which antigen-coated QdotBs were incubated with which
target antibody-spiked human serum samples. For control (no target
antibody) samples, human serum with no target antibodies was added
instead. The rest of the target antibody capture assay followed the
methods described above.

[0211]Photomasks of the desired microchannel pattern were prepared using
AutoCAD software (San Rafael, Calif.) and printed on a transparency by
the Photoplot Store (Colorado Springs, Colo.). The resolution of the
print was 1.59 μm (the distance between two pixels). Fabrication of
the masters began by spin coating a 15 μm thick layer of 2015 series
SU8 photoresist (MicroChem Corp., Newton, Mass.) on 3.5 inch diameter Si
wafers (Virginia Semiconductor, Fredericksburg, Va.) and prebaking the
samples. Each wafer then had the photomask laid on top of the
photoresist, ink surface down, and was exposed to 365 nm UV light at a
power density of 35 mW/cm2 for a duration of ˜4 seconds using a
SUSS MA6 mask aligner (SUSS MicroTec Inc., Waterbury Center, Vt.).
Following standard postbaking procedures, the wafers were immersed in SU8
Nanodeveloper (MicroChem Corp.) for ˜1 minute to dissolve away any
photoresist not exposed to the UV light. The masters were then washed
with isopropanol and dried with compressed N2 gas.

[0212]The polydimethylsiloxane (PDMS) prepolymer kits (RTV 615, General
Electric Silicones, Wilton, Conn.) used come in two parts; part A is the
prepolymer; part B contains a cross-linker. Prepolymer was mixed in a
10A:1B ratio. Masters were placed in pyrex Petri dishes and 22 g of
prepolymer was poured on top of each. The samples were then placed under
vacuum for ˜2 hrs to degas (remove bubbles from) the prepolymer. A
3-hour incubation followed in an oven set at 80° C. Once removed
from the oven, the cured PDMS slabs were peeled off the masters and
excess polymer around the outside of the microchannel pattern was
removed. A single master had patterns for two polymer microchannels. The
surfaces of the PDMS substrates and glass coverslips (170 μm thick,
VWR, Mississauga, ON) were then carefully cleaned using scotch tape. Both
PDMS substrates and glass coverslips were loaded into the chamber of a
plasma cleaner (Harrick Plasma, Ithaca, N.Y.) and exposed to oxygen
plasma for 1 min. Immediately after, the surfaces of the PDMS and
coverslips were brought into contact to irreversibly seal the two
substrates together. Double distilled water was dispensed into the
microchannels to keep the channel surfaces hydrophilic. Finally, small
pieces of glass were placed on top of the channel wells to keep the water
from evaporating, enabling long term storage of the samples.

[0213]Detection Experiments

[0214]First, QdotB complexes in 500 μL of TRIS wash buffer were
centrifuged at 4000 rpm for 3 minutes and aspirated. They were then
resuspended in 30 μL of double distilled water.

[0215]Microchannels were flushed with double distilled water once before
use, by filling the buffer and waste wells and applying suction at the
sample well using a custom tool. Fluid was removed from all wells prior
to the introduction of sample into the chip. 20 μL of sample were
loaded into the sample well, followed by 20 μL of double distilled
water into each of the buffer and waste wells. The microfluidic chip was
then aligned on the stage of an inverted epiflourescent microscope (IX71,
Olympus, Center Valley, Pa.) and immersion oil was applied to the lens of
a 60× objective (1.35 NA, Olympus). The objective lens was brought
into focus at the entrance of the sample well.

[0216]Electrodes were placed in each of the wells, leads connected to the
outputs of a voltage regulation circuit (see FIG. S2). The input of the
voltage regulation circuit was connected to a high voltage power supply
(CZE1000R, Spellman High Voltage Electronics Corp., Hauppauge, N.Y.),
which supplied 300V and 60 μA to the regulation circuit during a
typical experiment. The voltage ratio between the buffer and sample
channels was set at 1.8.

[0217]Once QdotB complexes started to flow into the microchannel 206 as
described above, the objective lens focus was moved to align with the
sample focusing stream located downstream from the intersection 230 of
the buffer channel 208 and sample channel 206. The objective lens 330 was
then used to focus a laser spot, measuring ˜8 μm in diameter and
using the 488 nm Ar laser 310 line from a multi-line, Ar/Kr gas laser
(COHERENT Inc., Santa Clara, Calif.) in TEM00 mode, on the ˜10
μm wide single-file sample stream 140. The laser power was set at a
constant 25 mW. A dichroic mirror (U-N41001, Olympus) and 500 nm longpass
emission filter 320 (7512, Chroma Technology Corp., Rockingham, Vt.) were
used to separate the excitation light 302 from the collected fluorescence
304. Fluorescence emission 304 was separated into spectral bands using
dirchroic mirrors (q555lp and 610dlp, Chroma Technology Corp.) and
bandpass filters 440a-d before being focused on the active areas of
solid-state photo detectors 410a-d (see FIG. 3A).

[0218]The spectra of the Qdots and bandpass filters used is displayed in
FIG. 6A. A comparison of the spectral peaks for the red, yellow and
orange QdotBs is displayed in FIG. 6B. For green range wavelengths, shown
as peaks 602d for the filter, and 604d for the raw fluorescence, a
500-540 nm bandpass filter 440a (HQ520/40, Chroma Technology Corp.) was
used with a bi-convex lens (LB1761-A, Thorlabs Inc., Newton, N.J.) to
focus these emissions on a PIN photodiode detector 410a (818 Series,
Newport Corp., Irvine, Calif.). This detector was connected to an optical
power meter (1830-C, Newport Corp.). Similarly, bandpass filters 440b and
440d (HQ575/30 and HQ630/60, Chroma Technology Corp.) and lenses
(LB1811-A and LA1027-A, respectively, Thorlabs Inc.) were used for two
avalanche photodiodes (C4777-01 and C5460 for yellow and red channels,
respectively, Hamamatsu Corp., Bridgewater, N.J.). The yellow range of
wavelengths are shown as peaks 602a for the filter, and 604a for the raw
fluorescence and the red range of wavelengths are shown as peaks 602b for
the filter, and 604b for the raw fluorescence. Voltage outputs for all
three detectors were connected to ports of a data acquisition card (NI
USB-6251, National Instruments, Austin, Tex.) that was relayed to a
computer and operated using Labview software (National Instruments).
Sampling at 1 kHz was used, though the capabilities of the data
acquisition card are on the order of 1 MS/s. for comparison, FIG. 6A also
shows the orange range of wavelengths as peaks 602c for the filter (see
above) and 604c for the raw Qdot spectrum. The spectra for the red 612b,
yellow 612a and orange 612c QdotB complexes are overlaid in FIG. 6B for
comparison purposes.

[0219]A typical experiment ran for 15 minutes, allowing the collection of
˜30 MB of data and ˜1000 detection events. After an
experiment, the QdotB complexes remaining in the sample channel were
collected and counted using a cell counter (Vi-Cell XR, Beckman-Coulter,
Fullerton, Calif.) and the microchannel was discarded using appropriate
disposal technniques. QdotB complex concentrations in the sample well
were 1.5×107/mL (9×106 standard deviation) during a typical
experiment.

[0220]Collection of Spectra Using a CCD Array Camera

[0221]The collection of spectra such as those shown in FIGS. 7A-C used
similar steps to those given above. Fluorescence emission, however, was
directed towards and focused on the entrance slit of a spectrograph
(Acton Research Inc., Acton, Mass.) using a 50.0 mm focal length lens
(LA1131-A, Thorlabs Inc.). Inside the spectrograph a grating with 150
grooves per mm was used to disperse the emission light by wavelength
before illuminating the pixels of a thermo-electrically cooled CCD camera
(7481-0002, Princeton Instruments, Trenton, N.J.). The selection of this
grating allowed inspection of the spectrum from ˜450-700 nm when
aligned for a central wavelength of 570 nm. The integration time of the
CCD array was 50 msec, set by a mechanical shutter, while typical readout
time was ˜250 msec. Data taken by the camera was collected and
analyzed using WinSpec software (Princeton Instruments). By collecting
multiple, successive spectra, it was possible to discriminate background
and detection signals from each other. The raw data for the HBV, HIV and
HCV samples are shown as 724a, 724b and 724c, respectively, with fitted
curves matched to the raw data as distinct fluorescent spectra 726a, 726b
and 726c, respectively. The respective fitted curves show yellow peaks
604a, red peaks 604b, which combine for orange peak 604c. The green peak
604d for the target fluorophore is also present.

[0222]Theory

[0223]Downstream of the sample and buffer channels, the channel undergoes
flow focusing. Flow focusing is an important aspect of the technology
since microbeads in the flow tend to non-specifically adsorb onto the
PDMS which can greatly affect the Qdot-barcode measurements. With flow
focusing, the Qdot-barcode interaction with the PDMS substrate is
minimized.

[0224]The size and shape of the channels in the microfluidic chip is
determined by the size of the beads and conjugates being detected. A 5
μm bead, for example, requires approximately 7-8 μm of space to
flow after functionalization and conjugation, therefore a focused flow
channel no larger 10 μm in width allows for regular flow of the beads,
while only permitting one beads to pass through the channel at a time.

[0225]The configuration of the channels is optimized to permit the focused
flow microbeads to travel past the detection point one at a time, while
maintaining the flow rate such that there is no clumping or
agglomeration. The configuration depends on several factors, including
the voltage applied to the focusing channel, the voltage applied to the
sample channel, and the length of the various channels.

is determined by the buffer and channel wall material, ε, ξ,
μ have a relationship with buffer solution's pH, temperature and other
characteristics. From Equation (1) above it is shown that the microbead
velocity has to be >0 to build a stable flow, which sets the criteria
for selecting the buffer.

[0229]The voltage ratio (not absolute voltage) of the focusing channel to
the sample channel (at the sample well) α=Uf/Ui is
subject to

αmin≦α≦σmax (2)

[0230]Where αmin and αmax are related to the length
of each part of the channel.

Lo , Li , l Lf are the length of the outlet, inlet and
focusing channel, respectively. As the focusing channel in the preferred
chip design has an L-shape, Lf is defined as the sum of Lf1 and
Lf2, the two arms of the channel.

[0231]Theoretically, there is no limitation for voltage applied to one of
the channels (focusing or sample). As shown in Equation (1), flow
velocity is proportional to the voltage, such that a higher voltage
results in a larger flow velocity. However, there is limitation for the
voltage ratio as shown in equation (2). Beyond this range, the flow
cannot be generated.

[0232]Thus, the ratio of the focused width, Wf of the sample flow, is
related to the width of the inlet channel according to the equation:

[0239]The formulas above are based on the assumption that all the channels
are straight, with no convergence and divergence, and the height is the
same for all branches. If the assumption is incorrect, the formula will
change, but will obey the same principles as outlined. In addition, there
is no limitation for the length of each branch, as different length
combinations will merely result in different widths for the focused
fluid.

[0240]Data Analysis

[0241]FIGS. 8A-C and show 10-second intervals of raw data collected during
experiments where large fluctuations in detector output voltages indicate
detection events in real time. For these experiments, a PIN photodiode
coupled to an optical power meter and amplifier was used to examine green
(500-540 nm) wavelengths 802a-c, corresponding to the target fluorophore
part of the spectrum, shown as 604d in FIG. 7A-C, while APDs were used
for yellow 804a-c and red channels 806a-c (550-590 nm and 600-650 nm,
respectively), which correspond to the BRM part of the spectrum shown as
604a-c in FIGS. 7A-C. Outputs from all three detectors were linked to a
computer using a data acquisition card and run using Labview software.
Since the speed that a barcode traverses the laser spot is inversely
proportional to the peak intensity measured by a detector, normalized
voltage peak values with respect to time were used for signal analysis.
For example, the green channel, which indicates target antibody
detection, used the metric G=∫.sup.peakV(t)dt/∫peakdt,
where V(t) is the voltage signal as a function of time, t. FIG. 9
provides a close-up of a series of peak to demonstrate the range of
voltages.

[0242]False positives are a common clinically encountered problem for
assays being performed at target molecule levels approaching the
detection sensitivity limit of the diagnostic. Therefore, assessing the
detection limit for the platform is important and serial dilution
sensitivity curves for HBV HBsAg, HCV NSP4 and HIV gp41 target
antibodies were prepared and compared to commercially available ELISA
kits. The detection algorithm first scanned the green channel for peaks
and then made sure appropriate peaks were also present in the yellow and
red channels before a detection event was confirmed. The values for
detection peaks are plotted in FIGS. 10A-C. Log curves 1000a, 1000b and
1000c are respectively fitted to data for HBV, HIV and HCV, with expanded
views 1002a, 1002b and 1002c respectively showing addition detail at the
tail end of the curves. For HBsAg antibodies, the detection sensitivity
limit was measured in the femtomolar (10-13-10-15M) range,
while the limits for NSP4 and gp41 were on the picomolar scale
(10-10-10-12M).

[0243]The required bead concentration is based on the need to measure the
single bead signal; high bead concentrations require higher speed
detectors and data acquisition systems. Bead to bead interactions become
a factor due to the small separation between beads, which will affect
flow. For low bead concentrations, it will take longer to generate enough
counts for statistical analysis (over 1000 in the current experiment).
The average bead concentration is 15×106 m/L with a standard
deviation of 9×106 m/L in the examples shown, taking
approximately 15 minutes to get >1000 counts. The range of acceptable
concentration is thus estimated between 15×107 m/L and
15×105 m/L, with a corresponding change in the time required
for count acquisition. The actual size of the beads can range from as a
little as 100 nm up to 5 μm.

[0244]A major benefit of using fluorescent barcodes is their multiplexing
detection capacity and the ability to apply it to pathogen detection.
FIGS. 11A-D, 12 and 13A-B show results of two (HBV and HIV) and three
(HBV, HCV and HIV) pathogen multiplexing experiments. The detector data
was analyzed by first indicating where green channel peaks were present,
and then classified as HBV, HCV or HIV detection events based on the
ratio of normalized values R/Y. FIGS. 11A-C show how histograms 1100a,
1100b, 1100c of detection events from HBV, HIV and HCV have clearly
distinguishable differences in the R/Y ratios, from low to medium to
high, respectively, with a comparison of all three in FIG. 11D. By using
this approach, it was possible to identify the different pathogen
detection events in the same sample, and when the target molecules
present during the assay were modified, the results accounted for this
change.

[0245]FIG. 12 shows a table of experiments used for FIGS. 13A and 13B).
FIG. 13A shows the results of 2 pathogen multiplexing experiments with
HBV and HIV. FIG. 13B shows the results of 3 pathogen multiplexing
experiments with HBV, HCV and HIV. These results show negligible
cross-reactivity for these three pathogen markers. Concentrations of HBV,
HIV, and HCV antibodies for this experiment were all 4.74×10-9 M.
The error bars shown represent one standard deviation.

[0246]The microfluidic detection system represents a successful
convergence of nano- and microtechnologies with molecular diagnostics
into a multiplexed infectious disease bioanalytical tool. Certain
modifications can be made to the system to adapt it for detection of
specific molecules or use with specific antibodies. Other modification
can be made to adjust the size and structure of the overall system
incorporating the microfluidic chip. For example, an LED or other
radiation emitting element may be used in place of the laser for the
purpose of exciting the molecules. Further developments and refinements,
not all of which will be readily obvious to those skilled in the art, may
present themselves.

[0247]While the above method has been presented in the context of a
quantum dot-based barcode the method is equally applicable to fluorescent
dyes and other types of luminescent particles.

[0248]This concludes the description of a presently preferred embodiment
of the invention.